RNAi Agents for Inhibiting Expression of Alpha-ENaC And Methods of Use

ABSTRACT

Described are RNAi agents, compositions that include RNAi agents, and methods for inhibition of an alpha-ENaC (SCNN1A) gene. The alpha-ENaC RNAi agents and RNAi agent conjugates disclosed herein inhibit the expression of an alpha-ENaC gene. Pharmaceutical compositions that include one or more alpha-ENaC RNAi agents, optionally with one or more additional therapeutics, are also described. Delivery of the described alpha-ENaC RNAi agents to epithelial cells, such as pulmonary epithelial cells, in vivo, provides for inhibition of alpha-ENaC gene expression and a reduction in ENaC activity, which can provide a therapeutic benefit to subjects, including human subjects.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/778,582, filed Jan. 31, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/028,006, filed Jul. 5, 2018, now U.S. Pat. No.10,590,416, which claims priority to U.S. Provisional Patent ApplicationSer. No. 62/679,549, filed on Jun. 1, 2018, U.S. Provisional PatentApplication Ser. No. 62/631,683, filed on Feb. 17, 2018, and U.S.Provisional Patent Application Ser. No. 62/529,132, filed on Jul. 6,2017, the contents of each of which are incorporated herein by referencein its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format and is hereby incorporated by reference in its entirety.The ASCII copy is named 30656-US2_SequenceListing and is 74 kb in size.

FIELD OF THE INVENTION

The present disclosure relates to RNA interference (RNAi) agents, e.g.,double stranded RNAi agents, for inhibition of alpha-ENaC geneexpression, compositions that include alpha-ENaC RNAi agents, andmethods of use thereof.

BACKGROUND

The vertebrate amiloride-sensitive epithelial sodium channel (“ENaC” or“amiloride-sensitive sodium channel”) is a member of the degenerin/ENaCchannel superfamily, characterized by two membrane-spanning domains,intracellular N- and C-termini, and a large extracellular loop which isa substrate for furin proteases. The channel is a heterotrimeric complexcomposed of three homologous subunits (alpha (α), beta (β), and gamma(γ)) encoded by three separate genes: SCNN1A (alpha), SCNN1B (beta), andSCNN1G (gamma). All three subunits are required for full channelactivity. A fourth subunit (delta (δ)) encoded by SCNN1D is expressed intestes and ovaries and may be able to functionally substitute for thealpha (α) subunit in those tissues.

ENaC is expressed on the apical membrane of epithelial cells,particularly in the lung, renal distal convoluted tubule,gastrointestinal (GI) tract, reproductive tract, and ocular surfaceepithelium in the eye. In these epithelia, ENaC channels mediate influxof extracellular sodium ions which are then actively transported fromthe cell by the basolateral sodium/potassium ATPase, establishing anosmotic gradient and causing epithelial luminal water to be absorbedinto the interstitium. In the kidney, ENaC mediates electrolyte balanceand blood pressure, and is the target of systemic small moleculediuretics such as amiloride. In the lung, airway epithelial ENaC plays akey role in the regulation of lung hydration and mucociliary clearance.

Type 1 pseudohypoaldosteronism (PHA) patients that carryloss-of-function mutations in SCNN1A, SCNN1B, or SCNN1G, produce excessairway surface liquid and have significantly higher mucociliaryclearance rates. Conversely, airway epithelial ENaC activity issignificantly elevated in cystic fibrosis (CF) patients of allgenotypes. Enhanced ENaC activity, together with reduced cystic fibrosistransmembrane conductance regulator (CFTR) chloride channel activity, isthe primary pathogenic mechanism that underlies airway dehydration andmucociliary stasis in CF lung disease patients.

Inhaled small molecule ENaC inhibitors have shown initial promise in thetreatment of CF, but their clinical development has been limited byshort duration of action in the lung and on-target toxicity(hyperkalemia) associated with inhibition of renal ENaC. (See, e.g.,O'Riordan et al., 27 J. Aerosol Med. & Pulmonary Drug Dev., 200-208(2014)).

Certain RNAi agents capable of inhibiting the expression of analpha-ENaC gene (i.e., SCNN1A) have been previously identified, such asthose disclosed in, for example, U.S. Pat. No. 7,718,632. However, thesequences and modifications of the alpha-ENaC RNAi agents disclosedherein differ from those previously disclosed or known in the art. Thealpha-ENaC RNAi agents disclosed herein provide for highly potent andefficient inhibition of the expression of an alpha-ENaC gene.

SUMMARY

There exists a need for novel RNA interference (RNAi) agents (termedRNAi agent, RNAi trigger, or trigger), e.g., double stranded RNAiagents, that are able to selectively and efficiently inhibit theexpression of the alpha-ENaC gene (i.e., SCNN1A). Further, there existsa need for compositions of novel alpha-ENaC-specific RNAi agents for thetreatment of diseases associated with enhanced ENaC activity.

In general, the present disclosure features alpha-ENaC gene-specificRNAi agents, compositions that include alpha-ENaC RNAi agents, andmethods for inhibiting expression of an alpha-ENaC gene in vitro and/orin vivo using the alpha-ENaC RNAi agents and compositions that includealpha-ENaC RNAi agents described herein. The alpha-ENaC RNAi agentsdescribed herein are able to selectively and efficiently decreaseexpression of an alpha-ENaC gene, and thereby reduce ENaC levels in asubject, reduce ENaC activity in a subject, or reduce both ENaC levelsand ENaC activity in a subject, e.g., a human or animal subject.

The described alpha-ENaC RNAi agents can be used in methods fortherapeutic treatment (including preventative or prophylactic treatment)of symptoms and diseases associated with enhanced or elevated ENaCactivity levels, including, but not limited to various respiratorydiseases such as cystic fibrosis, chronic bronchitis, chronicobstructive pulmonary disease (COPD), asthma, respiratory tractinfections, primary ciliary dyskinesia, and lung carcinoma cysticfibrosis. For example, in subjects suffering from cystic fibrosis (CF),increased ENaC activity is known to contribute to drying mucus in theairway and a reduced ability of the lung to clear toxins and infectiousagents. Further, it is also known that CF subjects that have inheritedpoorly functioning ENaC genes have shown milder lung disease, providingadditional evidence that inhibition ENaC levels may be beneficial forcertain patient populations. The described alpha-ENaC RNAi agents canalso be used, for example, for the therapeutic treatment (includingprophylactic or preventative treatment) of symptoms and diseasesassociated with enhanced or elevated ENaC activity levels in the ocularsurface epithelium, such as the conjunctival epithelium, including forthe treatment of ocular diseases and disorders such as dry eye syndrome.The alpha-ENaC RNAi agents disclosed herein can selectively reducealpha-ENaC expression, which can lead to a reduction in ENaC activity.The methods disclosed herein include the administration of one or morealpha-ENaC RNAi agents to a subject, e.g., a human or animal subject, byany suitable means known in the art, such as aerosol inhalation or drypowder inhalation, intranasal administration, intratrachealadministration, or oropharyngeal aspiration administration.

In one aspect, the disclosure features RNAi agents for inhibitingexpression of an alpha-ENaC gene, wherein the RNAi agent includes asense strand and an antisense strand. Also described herein arecompositions that include or consist of an RNAi agent capable ofinhibiting the expression of an alpha-ENaC gene, wherein the RNAi agentincludes or consists of a sense strand and an antisense strand, and thecomposition further comprises at least one pharmaceutically acceptableexcipient.

In another aspect, the disclosure features compositions that include oneor more of the disclosed alpha-ENaC RNAi agents that are able toselectively and efficiently decrease expression of the alpha-ENaC gene.The compositions that include one or more alpha-ENaC RNAi agentsdescribed herein can be administered to a subject, such as a human oranimal subject, for the treatment (including prophylactic treatment orinhibition) of symptoms and diseases associated with enhanced orelevated ENaC activity (also referred to herein as enhanced ENaC channelactivity levels or elevated ENaC channel activity levels).

Each alpha-ENaC RNAi agent disclosed herein includes a sense strand andan antisense strand. The sense strand and the antisense strand can bepartially, substantially, or fully complementary to each other. Thelength of the RNAi agent sense and antisense strands described hereineach can be 16 to 30 nucleotides. In some embodiments, the sense andantisense strands are independently 17 to 26 nucleotides in length. Thesense and antisense strands can be either the same length or differentlengths. In some embodiments, the sense and antisense strands areindependently 21 to 26 nucleotides in length. In some embodiments, thesense and antisense strands are independently 21 to 24 nucleotides inlength. In some embodiments, both the sense strand and the antisensestrand are 21 nucleotides in length. In some embodiments, the senseand/or antisense strands are independently 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The RNAi agentsdescribed herein, upon delivery to a cell expressing alpha-ENaC, inhibitthe expression of one or more alpha-ENaC genes in vivo or in vitro.

An alpha-ENaC RNAi agent described herein includes at least 16consecutive nucleotides that have at least 85% identity to a corestretch sequence (also referred to herein as a “core stretch” or “coresequence”) of the same number of nucleotides in an alpha-ENaC mRNA. Insome embodiments, this sense strand core stretch is 16, 17, 18, 19, 20,21, 22, or 23 nucleotides in length. In some embodiments, this sensestrand core stretch is 17 nucleotides in length. In some embodiments,this sense strand core stretch is 19 nucleotides in length.

An antisense strand of an alpha-ENaC RNAi agent described hereinincludes at least 16 consecutive nucleotides that have at least 85%complementarity to a core stretch of the same number of nucleotides inan alpha-ENaC mRNA and to the corresponding sense strand. In someembodiments, this antisense strand core stretch is 16, 17, 18, 19, 20,21, 22, or 23 nucleotides in length.

In some embodiments, the alpha-ENaC RNAi agents disclosed herein targeta portion of an alpha-ENaC gene having the sequence of any of thesequences disclosed in Table 1.

Examples of alpha-ENaC RNAi agent sense strands and antisense strandsthat can be used in an alpha-ENaC RNAi agent are provided in Tables 3and 4. Examples of alpha-ENaC RNAi agent duplexes are provided in Table5. Examples of 19-nucleotide core stretch sequences that may consist ofor may be included in the sense strands and antisense strands of certainalpha-ENaC RNAi agents disclosed herein, are provided in Table 2.

In another aspect, the disclosure features methods for deliveringalpha-ENaC RNAi agents to epithelial cells in a subject, such as amammal, in vivo. Also described herein are compositions for use in suchmethods. In some embodiments, disclosed herein are methods fordelivering alpha-ENaC RNAi agents to pulmonary epithelial cells in vivoto a subject. In some embodiments, disclosed herein are methods fordelivering alpha-ENaC RNAi agents to pulmonary epithelial cells of ahuman subject in vivo. The one or more alpha-ENaC RNAi agents can bedelivered to target cells or tissues using any oligonucleotide deliverytechnology known in the art. Nucleic acid delivery methods include, butare not limited to, by encapsulation in liposomes, by iontophoresis, orby incorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, proteinaceousvectors, or Dynamic Polyconjugates™ (DPCs) (see, for example WO2000/053722, WO 2008/022309, WO 2011/104169, and WO 2012/083185, each ofwhich is incorporated herein by reference).

In some embodiments, an alpha-ENaC RNAi agent is delivered to cells ortissues by covalently linking the RNAi agent to a targeting group. Insome embodiments, the targeting group can include a cell receptorligand, such as an integrin targeting ligand. Integrins are a family oftransmembrane receptors that facilitate cell-extracellular matrix (ECM)adhesion. In particular, integrin alpha-v-beta-6 (αvβ6) is anepithelial-specific integrin that is known to be a receptor for ECMproteins and the TGF-beta latency-associated peptide (LAP), and isexpressed in various cells and tissues. Integrin αvβ6 is known to behighly upregulated in injured pulmonary epithelium. In some embodiments,the alpha-ENaC RNAi agents described herein are linked to an integrintargeting ligand that has affinity for integrin αvβ6. As referred toherein, an “αvβ6 integrin targeting ligand” is a compound that hasaffinity for integrin αvβ6, which can be utilized as a ligand tofacilitate the targeting and delivery of an RNAi agent to which it isattached to the desired cells and/or tissues (i.e., to cells expressingintegrin αvβ6). In some embodiments, multiple αvβ6 integrin targetingligands or clusters of αvβ6 integrin targeting ligands are linked to analpha-ENaC RNAi agent. In some embodiments, the alpha-ENaC RNAiagent-αvβ6 integrin targeting ligand conjugates are selectivelyinternalized by lung epithelial cells, either through receptor-mediatedendocytosis or by other means.

Examples of targeting groups useful for delivering alpha-ENaC RNAiagents that include αvβ6 integrin targeting ligands are disclosed, forexample, in International Patent Application Publication No. WO2018/085415 and in U.S. Provisional Patent Application Nos. 62/580,398and 62/646,739, the contents of each of which are incorporated byreference herein in its entirety.

A targeting group can be linked to the 3′ or 5′ end of a sense strand oran antisense strand of an alpha-ENaC RNAi agent. In some embodiments, atargeting group is linked to the 3′ or 5′ end of the sense strand. Insome embodiments, a targeting group is linked to the 5′ end of the sensestrand. In some embodiments, a targeting group is linked internally to anucleotide on the sense strand and/or the antisense strand of the RNAiagent. In some embodiments, a targeting group is linked to the RNAiagent via a linker.

A targeting group, with or without a linker, can be attached to the 5′or 3′ end of any of the sense and/or antisense strands disclosed inTables 2, 3, and 4. A linker, with or without a targeting group, can beattached to the 5′ or 3′ end of any of the sense and/or antisensestrands disclosed in Tables 2, 3, and 4.

In another aspect, the disclosure features compositions that include oneor more alpha-ENaC RNAi agents that have the duplex structures disclosedin Table 5.

In some embodiments, described herein are compositions that include acombination or cocktail of at least two alpha-ENaC RNAi agents havingdifferent sequences. In some embodiments, the two or more alpha-ENaCRNAi agents are each separately and independently linked to targetinggroups. In some embodiments, the two or more alpha-ENaC RNAi agents areeach linked to targeting groups that include or consist of integrintargeting ligands. In some embodiments, the two or more alpha-ENaC RNAiagents are each linked to targeting groups that include or consist ofαvβ6 integrin targeting ligands.

In another aspect, the disclosure features methods for inhibitingalpha-ENaC gene expression in a subject, the methods includingadministering to the subject an amount of an alpha-ENaC RNAi agentcapable of inhibiting the expression of an alpha-ENaC gene, wherein thealpha-ENaC RNAi agent comprises a sense strand and an antisense strand.Also described herein are compositions for use in such methods.

In a further aspect, the disclosure features methods of treatment(including prophylactic or preventative treatment) of diseases orsymptoms caused by enhanced or elevated ENaC activity, the methodscomprising administering to a subject in need thereof an alpha-ENaC RNAiagent that includes an antisense strand comprising the sequence of anyof the sequences in Table 2 or Table 3. Also described herein arecompositions for use in such methods.

In some embodiments, the described alpha-ENaC RNAi agents are optionallycombined with one or more additional (i.e., second, third, etc.)therapeutics. A second therapeutic can be another alpha-ENaC RNAi agent(e.g., an alpha-ENaC RNAi agent that targets a different sequence withinthe alpha-ENaC gene). An additional therapeutic can also be a smallmolecule drug, antibody, antibody fragment, and/or aptamer. Thealpha-ENaC RNAi agents, with or without the one or more additionaltherapeutics, can be combined with one or more excipients to formpharmaceutical compositions.

In some embodiments, compositions for delivering an alpha-ENaC RNAiagent to an epithelial cell in vivo are described. In some embodiments,an alpha-ENaC RNAi agent is delivered without being conjugated to atargeting ligand or pharmacokinetic (PK) modulator (referred to as being“naked” or a “naked RNAi agent”). In some embodiments, an alpha-ENaCRNAi agent is conjugated to a targeting group, a linking group, a PKmodulator, and/or another non-nucleotide group. In some embodiments, analpha-ENaC RNAi agent is conjugated to a targeting group wherein thetargeting group includes an integrin targeting ligand. In someembodiment, the integrin targeting ligand is an αvβ6 integrin targetingligand. In some embodiments, a targeting group includes one or more αvβ6integrin targeting ligands.

In some embodiments, an alpha-ENaC RNAi agent is linked to one or morelinking groups or other non-nucleotide groups or compounds, such aspharmacokinetic modulators. In some embodiments, an alpha-ENaC RNAiagent is conjugated to a polyethylene glycol (PEG) moiety, or to ahydrophobic group having 12 or more carbon atoms, such as a cholesterolor palmitoyl group. In some embodiments, an alpha-ENaC RNAi agent islinked to one or more pharmacokinetic modulators selected fromcholesterol or cholesteryl derivatives, alkyl groups, alkenyl groups,alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, oraralkynyl groups, each of which may be linear, branched, cyclic, and/orsubstituted or unsubstituted. In some embodiments, the location ofattachment for these moieties is at the 5′ or 3′ end of the sensestrand, at the 2′ position of the ribose ring of any given nucleotide ofthe sense strand, and/or attached to the phosphate or phosphorothioatebackbone at any position of the sense strand.

In some embodiments, one or more of the described alpha-ENaC RNAi agentsare administered to a mammal in a pharmaceutically acceptable carrier ordiluent. In some embodiments, the mammal is a human.

The use of alpha-ENaC RNAi agents provides methods for therapeutic(including prophylactic) treatment of diseases or disorders associatedwith enhanced or elevated ENaC activity. The described alpha-ENaC RNAiagents are capable of inhibiting (e.g., inhibit) the expression ofalpha-ENaC. Alpha-ENaC RNAi agents can also be used to treat variousrespiratory diseases, including cystic fibrosis, chronic bronchitis,non-cystic fibrosis bronchiectasis, chronic obstructive pulmonarydisease (COPD), asthma, respiratory tract infections, primary ciliarydyskinesia, and lung carcinoma cystic fibrosis. Alpha-ENaC RNAi agentscan further be used to treat, for example, various ocular diseases anddisorders, such as dry eye. Such methods of treatment includeadministration of an alpha-ENaC RNAi agent to a human being or animalhaving elevated or enhanced ENaC activity levels. Described herein arecompositions for delivery of alpha-ENaC RNAi agents to pulmonaryepithelial cells. Furthermore, compositions for delivery of alpha-ENaCRNAi agents to cells, including renal epithelial cells and/or epithelialcells in the GI or reproductive tract and/or and ocular surfaceepithelial cells in the eye, in vivo, are generally described herein.

The pharmaceutical compositions including one or more alpha-ENaC RNAiagents can be administered in a number of ways depending upon whetherlocal or systemic treatment is desired. Administration can be, but isnot limited to, for example, intravenous, intraarterial, subcutaneous,intraperitoneal, subdermal (e.g., via an implanted device), andintraparenchymal administration. In some embodiments, the pharmaceuticalcompositions described herein are administered by inhalation (such asdry powder or aerosol inhalation), intranasal administration,intratracheal administration, or oropharyngeal aspirationadministration.

The described alpha-ENaC RNAi agents and/or compositions that includealpha-ENaC RNAi agents can be used in methods for therapeutic treatmentof disease or conditions caused by enhanced or elevated ENaC activitylevels. Such methods include administration of an alpha-ENaC RNAi agentas described herein to a subject, e.g., a human or animal subject.

In another aspect, the disclosure provides methods for the treatment(including prophylactic treatment) of a pathological state (such as acondition or disease) mediated at least in part by alpha-ENaCexpression, wherein the methods include administering to a subject atherapeutically effective amount of an RNAi agent that includes anantisense strand comprising the sequence of any of the sequences inTable 2 or Table 3.

In some embodiments, methods for inhibiting expression of an alpha-ENaCgene are disclosed herein, wherein the methods include administering toa cell an RNAi agent that includes an antisense strand comprising thesequence of any of the sequences in Table 2 or Table 3.

In some embodiments, methods for the treatment (including prophylactictreatment) of a pathological state mediated at least in part byalpha-ENaC expression are disclosed herein, wherein the methods includeadministering to a subject a therapeutically effective amount of an RNAiagent that includes a sense strand comprising the sequence of any of thesequences in Table 2 or Table 4.

In some embodiments, methods for inhibiting expression of an alpha-ENaCgene are disclosed herein, wherein the methods comprise administering toa cell an RNAi agent that includes a sense strand comprising thesequence of any of the sequences in Table 2 or Table 4.

In some embodiments, methods for the treatment (including prophylactictreatment) of a pathological state mediated at least in part byalpha-ENaC expression are disclosed herein, wherein the methods includeadministering to a subject a therapeutically effective amount of an RNAiagent that includes a sense strand comprising the sequence of any of thesequences in Table 4, and an antisense strand comprising the sequence ofany of the sequences in Table 3.

In some embodiments, methods for inhibiting expression of an alpha-ENaCgene are disclosed herein, wherein the methods include administering toa cell an RNAi agent that includes a sense strand comprising thesequence of any of the sequences in Table 4, and an antisense strandcomprising the sequence of any of the sequences in Table 3.

In some embodiments, methods of inhibiting expression of an alpha-ENaCgene are disclosed herein, wherein the methods include administering toa subject an alpha-ENaC RNAi agent that includes a sense strandconsisting of the nucleobase sequence of any of the sequences in Table4, and the antisense strand consisting of the nucleobase sequence of anyof the sequences in Table 3. In other embodiments, disclosed herein aremethods of inhibiting expression of an alpha-ENaC gene, wherein themethods include administering to a subject an alpha-ENaC RNAi agent thatincludes a sense strand consisting of the modified sequence of any ofthe modified sequences in Table 4, and the antisense strand consistingof the modified sequence of any of the modified sequences in Table 3.

In some embodiments, methods for inhibiting expression of an alpha-ENaCgene in a cell are disclosed herein, wherein the methods includeadministering one or more alpha-ENaC RNAi agents having a duplexstructure of one of the duplexes set forth in Table 5.

The alpha-ENaC RNAi agents disclosed herein are designed to targetspecific positions on an alpha-ENaC gene (SEQ ID NO: 1). As definedherein, an antisense strand sequence is designed to target an alpha-ENaCgene at a given position on the gene when the 5′ terminal nucleobase ofthe antisense strand is aligned with a position that is 19 nucleotidesdownstream (towards the 3′ end) from the position on the gene when basepairing to the gene. For example, as illustrated in Tables 1 and 2herein, an antisense strand sequence designed to target an alpha-ENaCgene at position 972 requires that when base pairing to the gene, the 5′terminal nucleobase of the antisense strand is aligned with position 990of the alpha-ENaC gene.

As provided herein, an alpha-ENaC RNAi agent does not require that thenucleobase at position 1 (5′→3′) of the antisense strand becomplementary to the gene, provided that there is at least 85%complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand andthe gene across a core stretch sequence of at least 16 consecutivenucleotides. For example, for an alpha-ENaC RNAi agent disclosed hereinthat is designed to target position 972 of an alpha-ENaC gene, the 5′terminal nucleobase of the antisense strand of the of the alpha-ENaCRNAi agent must be aligned with position 990 of the gene; however, the5′ terminal nucleobase of the antisense strand may be, but is notrequired to be, complementary to position 990 of an alpha-ENaC gene,provided that there is at least 85% complementarity (e.g., at least 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%complementarity) of the antisense strand and the gene across a corestretch sequence of at least 16 consecutive nucleotides.

As shown by, among other things, the various examples disclosed herein,the specific site of binding of the gene by the antisense strand of thealpha-ENaC RNAi agent (e.g., whether the alpha-ENaC RNAi agent isdesigned to target an alpha-ENaC gene at position 972, at position 1291,at position 1000, or at some other position) is a important factor forthe level of inhibition achieved by the alpha-ENaC RNAi agent.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGG (SEQ ID NO:3). In someembodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGG (SEQ ID NO:3), whereinall or substantially all of the nucleotides are modified nucleotides. Insome embodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGG (SEQ ID NO:3), whereinSEQ ID NO:3 is located at positions 1-21 (5′→3′) of the antisensestrand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a modified nucleotide sequence differing by no more than 1nucleotide from the nucleotide sequence (5′→3′)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg (SEQ ID NO:2), wherein a, c, g, andu represent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage, and wherein the sense strand is at leastsubstantially complementary to the antisense strand. As the person ofordinary skill in the art would clearly understand, the inclusion of aphosphorothioate linkage as shown in the modified nucleotide sequencesdisclosed herein replaces the phosphodiester linkage typically presentin oligonucleotides (see, e.g., FIGS. 12A through 12G showing allinternucleoside linkages).

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises the nucleotide sequence (5′→3′)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg (SEQ ID NO:2), wherein a, c, g, andu represent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage, and wherein the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesa sense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) CCUGUGCAACCAGAACAAAUA (SEQ ID NO:5). In someembodiments, an alpha-ENaC RNAi agent disclosed herein includes a sensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) CCUGUGCAACCAGAACAAAUA (SEQ ID NO:5), whereinall or substantially all of the nucleotides are modified nucleotides. Insome embodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) CCUGUGCAACCAGAACAAAUA (SEQ ID NO:5), whereinSEQ ID NO:5 is located at positions 1-21 (5′→3′) of the antisensestrand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesa sense strand that consists of, consists essentially of, or comprises amodified nucleotide sequence differing by no more than 1 nucleotide fromthe nucleotide sequence (5′→3′) cscugugcaAfCfCfagaacaaaua (SEQ ID NO:4),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage, and wherein the antisensestrand is at least substantially complementary to the sense strand. Insome embodiments, an alpha-ENaC RNAi agent disclosed herein includes asense strand that consists of, consists essentially of, or comprises themodified nucleotide sequence (5′→3′) cscugugcaAfCfCfagaacaaaua (SEQ IDNO:4), wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage, and wherein the antisensestrand is at least substantially complementary to the sense strand. Insome embodiments, one or more inverted abasic residues are added to the5′ end of the sense strand, to the 3′ end of the sense strand, or toboth the 5′ and the 3′ end of the sense strand of SEQ ID NO:4. In someembodiments, a targeting ligand, such as an αvβ6 integrin targetingligand, may be covalently linked to the 5′ end of the sense strand, tothe 3′ end of the sense strand, or to both the 5′ and the 3′ end of thesense strand of SEQ ID NO:4.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGG (SEQ ID NO:3) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) CCUGUGCAACCAGAACAAAUA (SEQ ID NO:5). In someembodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGG (SEQ ID NO:3), whereinall or substantially all of the nucleotides are modified nucleotides,and a sense strand that consists of, consists essentially of, orcomprises a nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) CCUGUGCAACCAGAACAAAUA (SEQ IDNO:5), wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises the modified nucleotide sequence (5′→3′)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg (SEQ ID NO:2), and a sense strandthat consists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) cscugugcaAfCfCfagaacaaaua (SEQ ID NO:4),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage. In some embodiments, analpha-ENaC RNAi agent disclosed herein includes an antisense strand thatconsists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg (SEQ IDNO:2), and a sense strand that consists of, consists essentially of, orcomprises the modified nucleotide sequence (5′→3′)cscugugcaAfCfCfagaacaaaua (SEQ ID NO:4), and wherein the sense strandfurther comprises an inverted abasic residue at the 3′ terminal end andan αvβ6 integrin targeting ligand covalently linked to the 5′ terminalend.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a nucleobase sequence that differs by 0 or 1 nucleobases fromthe nucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGC (SEQ ID NO:7). Insome embodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGC (SEQ ID NO:7), whereinall or substantially all of the nucleotides are modified nucleotides. Insome embodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGC (SEQ ID NO:7), whereinSEQ ID NO:7 is located at positions 1-21 (5′→3′) of the antisensestrand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a modified nucleotide sequence differing by no more than 1nucleotide from the modified nucleotide sequence (5′→3′)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc (SEQ ID NO:6), wherein a, c, g, andu represent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage, and wherein the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesa sense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) GCUGUGCAACCAGAACAAAUA (SEQ ID NO:9). In someembodiments, an alpha-ENaC RNAi agent disclosed herein includes a sensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) GCUGUGCAACCAGAACAAAUA (SEQ ID NO:9), whereinall or substantially all of the nucleotides are modified nucleotides. Insome embodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) GCUGUGCAACCAGAACAAAUA (SEQ ID NO:9), whereinSEQ ID NO:9 is located at positions 1-21 (5′→3′) of the antisensestrand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesa sense strand that consists of, consists essentially of, or comprises amodified nucleotide sequence that differs by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) gscugugcaAfCfCfagaacaaaua (SEQ IDNO:8), wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage, and wherein the antisensestrand is at least substantially complementary to the sense strand. Insome embodiments, one or more inverted abasic residues may be added tothe 5′ end of the sense strand, to the 3′ end of the sense strand, or toboth the 5′ and the 3′ end of the sense strand of SEQ ID NO:8. In someembodiments, a targeting ligand, such as an αvβ6 integrin targetingligand, may be covalently linked to the 5′ end of the sense strand, tothe 3′ end of the sense strand, or to both the 5′ and the 3′ end of thesense strand of SEQ ID NO:8.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGC (SEQ ID NO:7) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) GCUGUGCAACCAGAACAAAUA (SEQ ID NO:9). In someembodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACAGC (SEQ ID NO:7), whereinall or substantially all of the nucleotides are modified nucleotides,and a sense strand that consists of, consists essentially of, orcomprises a nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) GCUGUGCAACCAGAACAAAUA (SEQ IDNO:9), wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a modified nucleotide sequence differing by no more than 1nucleotide from the nucleotide sequence (5′→3′)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc (SEQ ID NO:6), and a sense strandthat consists of, consists essentially of, or comprises a modifiednucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) gscugugcaAfCfCfagaacaaaua (SEQ ID NO:8),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage. In some embodiments, analpha-ENaC RNAi agent disclosed herein includes an antisense strand thatconsists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc (SEQ IDNO:6), and a sense strand that consists of, consists essentially of, orcomprises the modified nucleotide sequence (5′→3′)gscugugcaAfCfCfagaacaaaua (SEQ ID NO:8), and wherein the sense strandfurther comprises an inverted abasic residue at the 3′ terminal end andan αvβ6 integrin targeting ligand covalently linked to the 5′ terminalend.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a modified nucleotide sequence differing by no more than 1nucleotide from the nucleotide sequence (5′→3′)cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg (SEQ ID NO:10), wherein a, c, g,and u represent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; s represents aphosphorothioate linkage, cPrpu represents a 5′-cyclopropylphosphonate-2′-O-methyl uridine (see Table 6), and wherein the sensestrand is at least substantially complementary to the antisense strand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a modified nucleotide sequence differing by no more than 1nucleotide from the nucleotide sequence (5′→3′)cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg (SEQ ID NO:10), and a sensestrand that consists of, consists essentially of, or comprises amodified nucleotide sequence differing by no more than 1 nucleotide fromthe nucleotide sequence (5′→3′) cscugugcaAfCfCfagaacaaaua (SEQ ID NO:4),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; srepresents a phosphorothioate linkage, and cPrpu represents a5′-cyclopropyl phosphonate-2′-O-methyl uridine (see Table 6). In someembodiments, an alpha-ENaC RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′→3′)cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg (SEQ ID NO:10), and a sensestrand that consists of, consists essentially of, or comprises themodified nucleotide sequence (5′→3′) cscugugcaAfCfCfagaacaaaua (SEQ IDNO:4), and wherein the sense strand further comprises an inverted abasicresidue at the 3′ terminal end and an αvβ6 integrin targeting ligandcovalently linked to the 5′ terminal end.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a nucleotide sequence that differs by 0 or 1 nucleotides fromone of the following nucleotide sequences (5′→3′):

(SEQ ID NO: 3) UAUUUGUUCUGGUUGCACAGG; (SEQ ID NO: 7)UAUUUGUUCUGGUUGCACAGC; (SEQ ID NO: 230) UGAUUUGUUCUGGUUGCACAG; or(SEQ ID NO: 254) AGAAGUCAUUCUGCUCUGCUU;wherein the alpha-ENaC RNAi agent further includes a sense strand thatis at least partially complementary to the antisense strand; and whereinthe all or substantially all of the nucleotides on both the antisensestrand and the sense strand are modified nucleotides.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a nucleotide sequence that differs by 0 or 1 nucleotides fromone of the following nucleotide sequences (5′→3′):

(SEQ ID NO: 3) UAUUUGUUCUGGUUGCACAGG; (SEQ ID NO: 7)UAUUUGUUCUGGUUGCACAGC; (SEQ ID NO: 230) UGAUUUGUUCUGGUUGCACAG; or(SEQ ID NO: 254) AGAAGUCAUUCUGCUCUGCUU;wherein the alpha-ENaC RNAi agent further includes a sense strand thatis at least partially complementary to the antisense strand; wherein theall or substantially all of the nucleotides on both the antisense strandand the sense strand are modified nucleotides; wherein the sense strandincludes an inverted abasic residue at the 3′ terminal end; and whereinan αvβ6 integrin targeting ligand is linked to at the 5′ terminal end ofthe sense strand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a nucleotide sequence that differs by 0 or 1 nucleotides fromone of the following nucleotide sequences (5′→3′):

(SEQ ID NO: 3) UAUUUGUUCUGGUUGCACAGG; (SEQ ID NO: 7)UAUUUGUUCUGGUUGCACAGC; (SEQ ID NO: 230) UGAUUUGUUCUGGUUGCACAG; or(SEQ ID NO: 254) AGAAGUCAUUCUGCUCUGCUU;wherein the alpha-ENaC RNAi agent further includes a sense strand thatis at least partially complementary to the antisense strand; wherein theall or substantially all of the nucleotides on both the antisense strandand the sense strand are modified nucleotides; wherein the sense strandincludes an inverted abasic residue at the 3′ terminal end; wherein anαvβ6 integrin targeting ligand is linked to at the 5′ terminal end ofthe sense strand; and wherein the respective antisense strand sequenceis located at positions 1-21 of the antisense strand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand and a sense strand, wherein the antisense strand andthe sense strand consist of, consist essentially of, or comprisenucleotide sequences that differ by 0 or 1 nucleotides from one of thefollowing nucleotide sequence (5′→3′) pairs:

(SEQ ID NO: 3) UAUUUGUUCUGGUUGCACAGG and (SEQ ID NO: 5)CCUGUGCAACCAGAACAAAUA; (SEQ ID NO: 7) UAUUUGUUCUGGUUGCACAGC and(SEQ ID NO: 9) GCUGUGCAACCAGAACAAAUA; (SEQ ID NO: 230)UGAUUUGUUCUGGUUGCACAG and (SEQ ID NO: 259) CUGUGCAACCAGAACAAAUCA; or(SEQ ID NO: 254) AGAAGUCAUUCUGCUCUGCUU and (SEQ ID NO: 289)GCAGAGCAGAAUGACUUCUUU;

-   -   wherein all or substantially all of the nucleotides on both the        antisense strand and the sense strand are modified nucleotides.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand and a sense strand, wherein the antisense strand andthe sense strand consist of, consist essentially of, or comprisenucleotide sequences that differ by 0 or 1 nucleotides from one of thefollowing nucleotide sequences (5′→3′) pairs:

(SEQ ID NO: 3) UAUUUGUUCUGGUUGCACAGG and (SEQ ID NO: 5)CCUGUGCAACCAGAACAAAUA; (SEQ ID NO: 7) UAUUUGUUCUGGUUGCACAGC and(SEQ ID NO: 9) GCUGUGCAACCAGAACAAAUA; (SEQ ID NO: 230)UGAUUUGUUCUGGUUGCACAG and (SEQ ID NO: 259) CUGUGCAACCAGAACAAAUCA; or(SEQ ID NO: 254) AGAAGUCAUUCUGCUCUGCUU and (SEQ ID NO: 289)GCAGAGCAGAAUGACUUCUUU;wherein all or substantially all of the nucleotides on both theantisense strand and the sense strand are modified nucleotides; whereinthe sense strand includes an inverted abasic residue at the 3′ terminalend; and wherein an αvβ6 integrin targeting ligand is linked to at the5′ terminal end of the sense strand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a modified nucleotide sequence that differs by 0 or 1nucleotides from one of the following nucleotide sequences (5′→3′):

(SEQ ID NO :2) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg; (SEQ ID NO: 6)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc; (SEQ ID NO: 10)cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg; (SEQ ID NO: 107)usGfsasUfuUfgUfuCfuGfgUfuGfcAfcAfsg;  or (SEQ ID NO: 152)asGfsasAfgUfcAfuUfcUfgCfuCfuGfcusu; wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; srepresents a phosphorothioate linkage, cPrpu represents a 5′-cyclopropylphosphonate-2′-O-methyl uridine (see Table 6); wherein the alpha-ENaCRNAi agent further includes the sense strand that is at least partiallycomplementary to the antisense strand; and wherein the all orsubstantially all of the nucleotides on the sense strand are modifiednucleotides.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand that consists of, consists essentially of, orcomprises a modified nucleotide sequence that differs by 0 or 1nucleotides from one of the following nucleotide sequences (5′→3′):

(SEQ ID NO: 2) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg; (SEQ ID NO: 6)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc; (SEQ ID NO: 10)cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg; (SEQ ID NO: 107)usGfsasUfuUfgUfuCfuGfgUfuGfcAfcAfsg;  or (SEQ ID NO: 152)asGfsasAfgUfcAfuUfcUfgCfuCfuGfcusu; wherein the alpha-ENaC RNAi agent further includes the sense strand thatis at least partially complementary to the antisense strand; wherein theall or substantially all of the nucleotides on the sense strand aremodified nucleotides; wherein the sense strand includes an invertedabasic residue at the 3′ terminal end; and wherein an αvβ6 integrintargeting ligand is linked to at the 5′ terminal end of the sensestrand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand and a sense strand that consists of, consistsessentially of, or comprise modified nucleotide sequences that differsby 0 or 1 nucleotides from one of the following nucleotide sequencepairs (5′→3′):

(SEQ ID NO: 2) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg and (SEQ ID NO: 4)cscugugcaAfCfCfagaacaaaua; (SEQ ID NO: 6)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc  and (SEQ ID NO: 8)gscugugcaAfCfCfagaacaaaua; (SEQ ID NO: 10)cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg  and (SEQ ID NO: 4)cscugugcaAfCfCfagaacaaaua; (SEQ ID NO: 107)usGfsasUfuUfgUfuCfuGfgUfuGfcAfcAfsg  and (SEQ ID NO: 293)csugugcaaCfCfAfgaacaaaucas;  or (SEQ ID NO: 152)asGfsasAfgUfcAfuUfcUfgCfuCfuGfcusu  and (SEQ ID NO: 294)gscagagCfAfGfaaugacuucuuu; wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; srepresents a phosphorothioate linkage, and cPrpu represents a5′-cyclopropyl phosphonate-2′-O-methyl uridine (see Table 6).

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand and a sense strand that consists of, consistsessentially of, or comprises one of the following nucleotide sequencepairs (5′→3′):

(SEQ ID NO: 2) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg  and (SEQ ID NO: 4)cscugugcaAfCfCfagaacaaaua; (SEQ ID NO: 6)usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc  and (SEQ ID NO: 8)gscugugcaAfCfCfagaacaaaua; (SEQ ID NO: 10)cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg  and (SEQ ID NO: 4)cscugugcaAfCfCfagaacaaaua; (SEQ ID NO: 107)usGfsasUfuUfgUfuCfuGfgUfuGfcAfcAfsg  and (SEQ ID NO: 293)csugugcaaCfCfAfgaacaaaucas;  or (SEQ ID NO: 152)asGfsasAfgUfcAfuUfcUfgCfuCfuGfcusu  and (SEQ ID NO: 294)gscagagCfAfGfaaugacuucuuu; wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; srepresents a phosphorothioate linkage, and cPrpu represents a5′-cyclopropyl phosphonate-2′-O-methyl uridine (see Table 6); whereinthe sense strand includes an inverted abasic residue at the 3′ terminalend; and wherein an αvβ6 integrin targeting ligand is linked to at the5′ terminal end of the sense strand.

In some embodiments, an alpha-ENaC RNAi agent disclosed herein includesan antisense strand comprises a nucleobase sequence that differs by 0 or1 nucleobases from the nucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACA(SEQ ID NO:21). In some embodiments, an alpha-ENaC RNAi agent disclosedherein includes an antisense strand that comprises a nucleotide sequencediffering by no more than 1 nucleotide from the nucleotide sequence(5′→3′) UAUUUGUUCUGGUUGCACA (SEQ ID NO:21), wherein all or substantiallyall of the nucleotides are modified nucleotides. In some embodiments, analpha-ENaC RNAi agent disclosed herein includes an antisense strand thatcomprises a nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UAUUUGUUCUGGUUGCACA (SEQ ID NO:21), whereinSEQ ID NO:21 is located at positions 1-19 (5′→3′) of the antisensestrand.

As used herein, the terms “oligonucleotide” and “polynucleotide” mean apolymer of linked nucleosides each of which can be independentlymodified or unmodified.

As used herein, an “RNAi agent” (also referred to as an “RNAi trigger”)means a composition that contains an RNA or RNA-like (e.g., chemicallymodified RNA) oligonucleotide molecule that is capable of degrading orinhibiting (e.g., degrades or inhibits under appropriate conditions)translation of messenger RNA (mRNA) transcripts of a target mRNA in asequence specific manner. As used herein, RNAi agents may operatethrough the RNA interference mechanism (i.e., inducing RNA interferencethrough interaction with the RNA interference pathway machinery(RNA-induced silencing complex or RISC) of mammalian cells), or by anyalternative mechanism(s) or pathway(s). While it is believed that RNAiagents, as that term is used herein, operate primarily through the RNAinterference mechanism, the disclosed RNAi agents are not bound by orlimited to any particular pathway or mechanism of action. RNAi agentsdisclosed herein are comprised of a sense strand and an antisensestrand, and include, but are not limited to: short interfering RNAs(siRNAs), double stranded RNAs (dsRNA), micro RNAs (miRNAs), shorthairpin RNAs (shRNA), and dicer substrates. The antisense strand of theRNAi agents described herein is at least partially complementary to themRNA being targeted (i.e. alpha-ENaC mRNA). RNAi agents can include oneor more modified nucleotides and/or one or more non-phosphodiesterlinkages.

As used herein, the terms “silence,” “reduce,” “inhibit,”“down-regulate,” or “knockdown” when referring to expression of a givengene, mean that the expression of the gene, as measured by the level ofRNA transcribed from the gene or the level of polypeptide, protein, orprotein subunit translated from the mRNA in a cell, group of cells,tissue, organ, or subject in which the gene is transcribed, is reducedwhen the cell, group of cells, tissue, organ, or subject is treated withthe RNAi agents described herein as compared to a second cell, group ofcells, tissue, organ, or subject that has not or have not been sotreated.

As used herein, the terms “sequence” and “nucleotide sequence” mean asuccession or order of nucleobases or nucleotides, described with asuccession of letters using standard nomenclature.

As used herein, a “base,” “nucleotide base,” or “nucleobase,” is aheterocyclic pyrimidine or purine compound that is a component of anucleotide, and includes the primary purine bases adenine and guanine,and the primary pyrimidine bases cytosine, thymine, and uracil. Anucleobase may further be modified to include, without limitation,universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases. (See, e.g., Modified Nucleosides inBiochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH,2008). The synthesis of such modified nucleobases (includingphosphoramidite compounds that include modified nucleobases) is known inthe art.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleobase or nucleotidesequence (e.g., RNAi agent sense strand or targeted mRNA) in relation toa second nucleobase or nucleotide sequence (e.g., RNAi agent antisensestrand or a single-stranded antisense oligonucleotide), means theability of an oligonucleotide or polynucleotide including the firstnucleotide sequence to hybridize (form base pair hydrogen bonds undermammalian physiological conditions (or similar conditions in vitro)) andform a duplex or double helical structure under certain standardconditions with an oligonucleotide or polynucleotide including thesecond nucleotide sequence. Complementary sequences include Watson-Crickbase pairs or non-Watson-Crick base pairs and include natural ormodified nucleotides or nucleotide mimics, at least to the extent thatthe above hybridization requirements are fulfilled. Sequence identity orcomplementarity is independent of modification. For example, a and Af,as defined herein, are complementary to U (or T) and identical to A forthe purposes of determining identity or complementarity.

As used herein, “perfectly complementary” or “fully complementary” meansthat in a hybridized pair of nucleobase or nucleotide sequencemolecules, all (100%) of the bases in a contiguous sequence of a firstoligonucleotide will hybridize with the same number of bases in acontiguous sequence of a second oligonucleotide. The contiguous sequencemay comprise all or a part of a first or second nucleotide sequence.

As used herein, “partially complementary” means that in a hybridizedpair of nucleobase or nucleotide sequence molecules, at least 70%, butnot all, of the bases in a contiguous sequence of a firstoligonucleotide will hybridize with the same number of bases in acontiguous sequence of a second oligonucleotide. The contiguous sequencemay comprise all or a part of a first or second nucleotide sequence.

As used herein, “substantially complementary” means that in a hybridizedpair of nucleobase or nucleotide sequence molecules, at least 85%, butnot all, of the bases in a contiguous sequence of a firstoligonucleotide will hybridize with the same number of bases in acontiguous sequence of a second oligonucleotide. The contiguous sequencemay comprise all or a part of a first or second nucleotide sequence.

As used herein, the terms “complementary,” “fully complementary,”“partially complementary,” and “substantially complementary” are usedwith respect to the nucleobase or nucleotide matching between the sensestrand and the antisense strand of an RNAi agent, or between theantisense strand of an RNAi agent and a sequence of an alpha-ENaC mRNA.

As used herein, the terms “substantially identical” or “substantialidentity,” as applied to a nucleic acid sequence means that a nucleotidesequence (or a portion of a nucleotide sequence) has at least about 85%sequence identity or more, e.g., at least 90%, at least 95%, or at least99% identity, compared to a reference sequence. Percentage of sequenceidentity is determined by comparing two optimally aligned sequences overa comparison window. The percentage is calculated by determining thenumber of positions at which the same type of nucleic acid base occursin both sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. The inventions disclosed hereinencompass nucleotide sequences substantially identical to thosedisclosed herein.

As used herein, the terms “treat,” “treatment,” and the like, mean themethods or steps taken to provide relief from or alleviation of thenumber, severity, and/or frequency of one or more symptoms of a diseasein a subject. As used herein, “treat” and “treatment” may includepreventative treatment, management, prophylactic treatment, and/orinhibition or reduction of the number, severity, and/or frequency of oneor more symptoms of a disease in a subject.

As used herein, the phrase “introducing into a cell,” when referring toan RNAi agent, means functionally delivering the RNAi agent into a cell.The phrase “functional delivery,” means delivering the RNAi agent to thecell in a manner that enables the RNAi agent to have the expectedbiological activity, e.g., sequence-specific inhibition of geneexpression.

Unless stated otherwise, use of the symbol

as used herein means that any group or groups may be linked thereto thatis in accordance with the scope of the inventions described herein.

As used herein, the term “isomers” refers to compounds that haveidentical molecular formulae, but that differ in the nature or thesequence of bonding of their atoms or in the arrangement of their atomsin space. Isomers that differ in the arrangement of their atoms in spaceare termed “stereoisomers.” Stereoisomers that are not mirror images ofone another are termed “diastereoisomers,” and stereoisomers that arenon-superimposable mirror images are termed “enantiomers,” or sometimesoptical isomers. A carbon atom bonded to four non-identical substituentsis termed a “chiral center.”

As used herein, unless specifically identified in a structure as havinga particular conformation, for each structure in which asymmetriccenters are present and thus give rise to enantiomers, diastereomers, orother stereoisomeric configurations, each structure disclosed herein isintended to represent all such possible isomers, including theiroptically pure and racemic forms. For example, the structures disclosedherein are intended to cover mixtures of diastereomers as well as singlestereoisomers.

As used in a claim herein, the phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. When used in aclaim herein, the phrase “consisting essentially of” limits the scope ofa claim to the specified materials or steps and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

The person of ordinary skill in the art would readily understand andappreciate that the compounds and compositions disclosed herein may havecertain atoms (e.g., N, O, or S atoms) in a protonated or deprotonatedstate, depending upon the environment in which the compound orcomposition is placed. Accordingly, as used herein, the structuresdisclosed herein envisage that certain functional groups, such as, forexample, OH, SH, or NH, may be protonated or deprotonated. Thedisclosure herein is intended to cover the disclosed compounds andcompositions regardless of their state of protonation based on theenvironment (such as pH), as would be readily understood by the personof ordinary skill in the art.

As used herein, the term “linked” or “conjugated” when referring to theconnection between two compounds or molecules means that two compoundsor molecules are joined by a covalent bond. Unless stated, the terms“linked” and “conjugated” as used herein may refer to the connectionbetween a first compound and a second compound either with or withoutany intervening atoms or groups of atoms.

As used herein, the term “including” is used to herein mean, and is usedinterchangeably with, the phrase “including but not limited to.” Theterm “or” is used herein to mean, and is used interchangeably with, theterm “and/or,” unless the context clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other objects, features, aspects, and advantages of the invention willbe apparent from the following detailed description, accompanyingfigures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Histogram showing relative expression of mouse whole lungalpha-ENaC expression after administration of various alpha-ENaC RNAiagents compared to vehicle control.

FIG. 2. Histogram showing relative expression of mouse whole lungalpha-ENaC expression after administration of alpha-ENaC RNAi agentsAD04025 and AD04858 compared to vehicle control.

FIG. 3. Graph showing relative expression of rat whole lung alpha-ENaCexpression of alpha-ENaC RNAi agents AD04025 and AD04025-conjugate(i.e., AD04025 conjugated to a peptide-based αvβ6 epithelial celltargeting ligand).

FIG. 4. Chemical structure representation of the tridentate αvβ6epithelial cell targeting ligand referred to herein as Tri-SM2.

FIG. 5. Chemical structure representation of the tridentate αvβ6epithelial cell targeting ligand referred to herein as Tri-SM1.

FIG. 6. Chemical structure representation of the tridentate αvβ6epithelial cell targeting ligand referred to herein as Tri-SM6.1.

FIG. 7. Chemical structure representation of the tridentate αvβ6epithelial cell targeting ligand referred to herein as Tri-SM9.

FIG. 8. Chemical structure representation of the tridentate αvβ6epithelial cell targeting ligand referred to herein as Tri-SM6.

FIG. 9. Chemical structure representation of the tridentate αvβ6epithelial cell targeting ligand referred to herein as Tri-SM8.

FIG. 10. Chemical structure representation of the tridentate αvβ6epithelial cell targeting ligand referred to herein as Tri-SM10.

FIG. 11. Chemical structure representation of the tridentate αvβ6epithelial cell targeting ligand referred to herein as Tri-SM11.

FIG. 12A. Schematic diagram of the modified sense and antisense strandsof alpha-ENaC RNAi agent AD05453 (see Tables 3-5), shown with an aminogroup on the 5′ terminal end of the sense strand for facilitating thelinkage to targeting ligands.

-   -   The following abbreviations are used in FIGS. 12A to 12G: a, c,        g, and u are 2′-O-methyl modified nucleotides; Af, Cf, Gf, and        Uf are 2′-fluoro modified nucleotides; p is a phosphodiester        linkage; s is a phosphorothioate linkage; invAb is an inverted        abasic residue; cPrp is a 5′ terminal cyclopropyl phosphonate        group (see Table 6); NH2-C6 is a C₆ amino group (see Table 6);        and TriAlk14 is a tri-alkyne linker having the structure        depicted herein (see Table 6).

FIG. 12B. Schematic diagram of the modified sense and antisense strandsof alpha-ENaC RNAi agent AD05924 (see Tables 3-5), shown functionalizedwith a tri-alkyne group on the 5′ terminal end of the sense strand forfacilitating the linkage to targeting ligands. As described herein,AD05453 and AD05924 have the same modified nucleotide sequences, andrepresent alternative approaches to synthesizing an alpha ENaC-RNAiagent conjugate disclosed herein.

FIG. 12C. Schematic diagram of the modified sense and antisense strandsof alpha-ENaC RNAi agent AD05625 (see Tables 3-5), shown functionalizedwith an amino group on the 5′ terminal end of the sense strand forfacilitating the linkage to targeting ligands.

FIG. 12D. Schematic diagram of the modified sense and antisense strandsof alpha-ENaC RNAi agent AD05347 (see Tables 3-5), shown functionalizedwith an amino group on the 5′ terminal end of the sense strand forfacilitating the linkage to targeting ligands.

FIG. 12E. Schematic diagram of the modified sense and antisense strandsof alpha-ENaC RNAi agent AD05831 (see Tables 3-5), shown functionalizedwith an amino group on the 5′ terminal end of the sense strand forfacilitating the linkage to targeting ligands.

FIG. 12F. Schematic diagram of the modified sense and antisense strandsof alpha-ENaC RNAi agent AD05833 (see Tables 3-5), shown functionalizedwith an amino group on the 5′ terminal end of the sense strand forfacilitating the linkage to targeting ligands.

FIG. 12G. Schematic diagram of the modified sense and antisense strandsof both alpha-ENaC RNAi agent AD05453 and alpha-ENaC RNAi agent AD05924(see Tables 3-5), wherein X represents a tridentate αvβ6 integrintargeting ligand (including any linkers).

FIG. 12H. Schematic diagram of an example tridentate αvβ6 integrintargeting ligand-RNAi agent conjugate described herein, wherein atridentate αvβ6 integrin targeting ligand is conjugated to the 5′terminal end of the sense strand. As shown therein, each αvβ6 representsan αvβ6 integrin targeting compound.

FIG. 13A to 13D. Chemical structure representation of alpha-ENaC RNAiagent AD05453, including an NH2-C6 terminal amino group, shown as asodium salt.

FIG. 14A to 14D. Chemical structure representation of alpha-ENaC RNAiagent AD05924, including a tri-alkyne functionalized linker group(TriAlk14), shown as a sodium salt.

FIG. 15A to 15E. Chemical structure representation of alpha-ENaC RNAiagent AD05453, shown conjugated to Tri-SM6.1, as a sodium salt. Asdiscussed herein, the same chemical structure can be synthesized using atri-alkyne functionalized linker group (TriAlk14), which can be addedthrough phosphoramidite synthesis, as set forth in the modified sensestrand nucleotide sequence for alpha-ENaC RNAi agent AD05924 (i.e.,AM07807-SS in Table 4).

FIG. 16A to 16D. Chemical structure representation of alpha-ENaC RNAiagent AD05453, including a NH2-C6 terminal functionalized amino group,shown as a free acid.

DETAILED DESCRIPTION

RNAi Agents

Described herein are RNAi agents for inhibiting expression of thealpha-ENaC (i.e., SCNN1A) gene (referred to herein as alpha-ENaC RNAiagents or alpha-ENaC RNAi triggers). Each alpha-ENaC RNAi agentcomprises a sense strand and an antisense strand. The sense strand andthe antisense strand each can be 16 to 30 nucleotides in length. In someembodiments, the sense and antisense strands each can be 17 to 26nucleotides in length. The sense and antisense strands can be either thesame length or they can be different lengths. In some embodiments, thesense and antisense strands are each independently 17 to 26 nucleotidesin length. In some embodiments, the sense and antisense strands are eachindependently 17-21 nucleotides in length. In some embodiments, both thesense and antisense strands are each 21-26 nucleotides in length. Insome embodiments, the sense and antisense strands are each 21-24nucleotides in length. In some embodiments, the sense strand is about 19nucleotides in length while the antisense strand is about 21 nucleotidesin length. In some embodiments, the sense strand is about 21 nucleotidesin length while the antisense strand is about 23 nucleotides in length.In some embodiments, both the sense and antisense strands are each 21nucleotides in length. In some embodiments, the RNAi agent sense andantisense strands are each independently 16, 17, 18, 19, 20, 21, 22, 23,24, 25, or 26 nucleotides in length. In some embodiments, a doublestranded RNAi agent has a duplex length of about 16, 17, 18, 19, 20, 21,22, 23 or 24 nucleotides.

In some embodiments, the region of perfect, substantial, or partialcomplementarity between the sense strand and the antisense strand is16-26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotidesin length and occurs at or near the 5′ end of the antisense strand(e.g., this region may be separated from the 5′ end of the antisensestrand by 0, 1, 2, 3, or 4 nucleotides that are not perfectly,substantially, or partially complementary).

The sense strand and antisense strand each contain a core stretch (alsoreferred to herein as a “core sequence” or a “core stretch sequence”))that is 16 to 23 nucleotides in length. An antisense strand core stretchis 100% (perfectly) complementary or at least 85% (substantially)complementary to a nucleotide sequence (sometimes referred to, e.g., asa target sequence) present in the alpha-ENaC target. A sense strand corestretch is 100% (perfectly) complementary or at least 85%(substantially) complementary to a core stretch in the antisense strand,and thus the sense strand core stretch is typically perfectly identicalor at least 85% identical to a nucleotide sequence (target sequence)present in the alpha-ENaC mRNA target. A sense strand core stretch canbe the same length as a corresponding antisense core stretch or it canbe a different length. In some embodiments, the antisense strand corestretch is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. Insome embodiments, the sense strand core stretch is 16, 17, 18, 19, 20,21, 22, or 23 nucleotides in length.

Examples of nucleotide sequences used in forming alpha-ENaC RNAi agentsare provided in Tables 2, 3, and 4. Examples of RNAi agent duplexes,that include the sense strand and antisense strand nucleotide sequencesin Tables 2, 3, and 4, are shown in Table 5.

The alpha-ENaC RNAi agent sense and antisense strands anneal to form aduplex. A sense strand and an antisense strand of an alpha-ENaC RNAiagent can be partially, substantially, or fully complementary to eachother. Within the complementary duplex region, the sense strand corestretch sequence is at least 85% complementary or 100% complementary tothe antisense core stretch sequence. In some embodiments, the sensestrand core stretch sequence contains a sequence of at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, or at least 23 nucleotides that is at least 85% or 100%complementary to a corresponding 16, 17, 18, 19, 20, 21, 22, or 23nucleotide sequence of the antisense strand core stretch sequence (i.e.,the sense and antisense core stretch sequences of an alpha-ENaC RNAiagent have a region of at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, or at least 23 nucleotidesthat is at least 85% base paired or 100% base paired.) In someembodiments, the antisense strand of an alpha-ENaC RNAi agent disclosedherein differs by 0, 1, 2, or 3 nucleotides from any of the antisensestrand sequences in Table 2 or Table 3. In some embodiments, the sensestrand of an alpha-ENaC RNAi agent disclosed herein differs by 0, 1, 2,or 3 nucleotides from any of the sense strand sequences in Table 2 orTable 4.

The sense strand and/or the antisense strand can optionally andindependently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides(extension) at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of thecore stretch sequences. The antisense strand additional nucleotides, ifpresent, may or may not be complementary to the corresponding sequencein the alpha-ENaC mRNA. The sense strand additional nucleotides, ifpresent, may or may not be identical to the corresponding sequence inthe alpha-ENaC mRNA. The antisense strand additional nucleotides, ifpresent, may or may not be complementary to the corresponding sensestrand's additional nucleotides, if present.

As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotidesat the 5′ and/or 3′ end of the sense strand core stretch sequence and/orantisense strand core stretch sequence. The extension nucleotides on asense strand may or may not be complementary to nucleotides, either corestretch sequence nucleotides or extension nucleotides, in thecorresponding antisense strand. Conversely, the extension nucleotides onan antisense strand may or may not be complementary to nucleotides,either core stretch nucleotides or extension nucleotides, in thecorresponding sense strand. In some embodiments, both the sense strandand the antisense strand of an RNAi agent contain 3′ and 5′ extensions.In some embodiments, one or more of the 3′ extension nucleotides of onestrand base pairs with one or more 5′ extension nucleotides of the otherstrand. In other embodiments, one or more of 3′ extension nucleotides ofone strand do not base pair with one or more 5′ extension nucleotides ofthe other strand. In some embodiments, an alpha-ENaC RNAi agent has anantisense strand having a 3′ extension and a sense strand having a 5′extension. In some embodiments, the extension nucleotide(s) are unpairedand form an overhang. As used herein, an “overhang” refers to a stretchof one or more unpaired nucleotides located at a terminal end of eitherthe sense strand or the antisense strand that does not form part of thehybridized or duplexed portion of an RNAi agent disclosed herein.

In some embodiments, an alpha-ENaC RNAi agent comprises an antisensestrand having a 3′ extension of 1, 2, 3, 4, 5, or 6 nucleotides inlength. In other embodiments, an alpha-ENaC RNAi agent comprises anantisense strand having a 3′ extension of 1, 2, or 3 nucleotides inlength. In some embodiments, one or more of the antisense strandextension nucleotides comprise uracil or thymidine nucleotides ornucleotides that are complementary to the corresponding alpha-ENaC mRNAsequence.

In some embodiments, the 3′ end of the antisense strand can includeadditional abasic residues (Ab). An “abasic residue” or “abasic site” isa nucleotide or nucleoside that lacks a nucleobase at the 1′ position ofthe sugar moiety. (See, e.g., U.S. Pat. No. 5,998,203). In someembodiments, Ab or AbAb can be added to the 3′ end of the antisensestrand. In some embodiments, abasic residue(s) can be added as invertedabasic residues (invAb) (see Table 6). (See, e.g., F. Czauderna, NucleicAcids Res., 2003, 31(11), 2705-16).

In some embodiments, the sense strand or the antisense strand mayinclude a “terminal cap,” which as used herein is a non-nucleotidecompound or other moiety that can be incorporated at one or more terminiof a strand of an RNAi agent disclosed herein, and can provide the RNAiagent, in some instances, with certain beneficial properties, such as,for example, protection against exonuclease degradation. Terminal capsare generally known in the art, and include inverted abasic residues, aswell as carbon chains such as a terminal C₃, C₆, or C₁₂ group. In someembodiments, a terminal cap is present at either the 5′ terminal end,the 3′ terminal end, or both the 5′ and 3′ terminal ends of the sensestrand.

In some embodiments, an alpha-ENaC RNAi agent comprises a sense strandhaving a 3′ extension of 1, 2, 3, 4, or 5 nucleotides in length. In someembodiments, one or more of the sense strand extension nucleotidescomprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide,or nucleotides that correspond to nucleotides in the alpha-ENaC mRNAsequence. In some embodiments, the 3′ sense strand extension includes orconsists of one of the following sequences, but is not limited to: T,UT, TT, UU, UUT, TTT, or TTTT (each listed 5′ to 3′).

In some embodiments, the 3′ end of the sense strand may includeadditional abasic residues. In some embodiments, UUAb, UAb, or Ab areadded to the 3′ end of the sense strand.

In some embodiments, one or more inverted abasic residues (invAb) areadded to the 3′ end of the sense strand. In some embodiments, one ormore inverted abasic residues or inverted abasic sites are insertedbetween the targeting ligand and the nucleobase sequence of the sensestrand of the RNAi agent. In some embodiments, the inclusion of one ormore inverted abasic residues or inverted abasic sites at or near theterminal end or terminal ends of the sense strand of an RNAi agentallows for enhanced activity or other desired properties of an RNAiagent.

In some embodiments, an alpha-ENaC RNAi agent comprises a sense strandhaving a 5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. Insome embodiments, one or more of the sense strand extension nucleotidescomprise uracil or adenosine nucleotides or nucleotides that correspondto nucleotides in the alpha-ENaC mRNA sequence. In some embodiments, thesense strand 5′ extension is one of the following sequences, but is notlimited to: CA, AUAGGC, AUAGG, AUAG, AUA, A, AA, AC, GCA, GGCA, GGC,UAUCA, UAUC, UCA, UAU, U, UU (each listed 5′ to 3′). A sense strand canhave a 3′ extension and/or a 5′ extension.

In some embodiments, the 5′ end of the sense strand can include one ormore additional abasic residues (e.g., (Ab) or (AbAb)). In someembodiments, one or more inverted abasic residues (invAb) are added tothe 5′ end of the sense strand. In some embodiments, one or moreinverted abasic residues can be inserted between the targeting ligandand the nucleobase sequence of the sense strand of the RNAi agent. Insome embodiments, the inclusion of one or more inverted abasic residuesat or near the terminal end or terminal ends of the sense strand of anRNAi agent may allow for enhanced activity or other desired propertiesof an RNAi agent. In some embodiments, an abasic (deoxyribose) residuecan be replaced with a ribitol (abasic ribose) residue.

In some embodiments, the 3′ end of the antisense strand core stretchsequence, or the 3′ end of the antisense strand sequence, may include aninverted abasic residue (invAb (see Table 6)).

Examples of sequences used in forming alpha-ENaC RNAi agents areprovided in Tables 2, 3, and 4. In some embodiments, an alpha-ENaC RNAiagent antisense strand includes a sequence of any of the sequences inTables 2 or 3. In some embodiments, an alpha-ENaC RNAi agent antisensestrand includes the sequence of nucleotides (from 5′ end→3′ end) 1-17,2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22,1-23, 2-23, 1-24, or 2-24, of any of the sequences in Table 2 or Table3. In certain embodiments, an alpha-ENaC RNAi agent antisense strandcomprises or consists of a modified sequence of any one of the modifiedsequences in Table 3. In some embodiments, an alpha-ENaC RNAi agentsense strand includes the sequence of any of the sequences in Tables 2or 4. In some embodiments, an alpha-ENaC RNAi agent sense strandincludes the sequence of nucleotides (from 5′ end→3′ end) 1-18, 1-19,1-20, 1-21, 1-22, 1-23, 1-24, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 3-20,3-21, 3-22, 3-23, 3-24, 4-21, 4-22, 4-23, 4-24, 5-22, 5-23, or 5-24, ofany of the sequences in Tables 2 or 4. In certain embodiments, analpha-ENaC RNAi agent sense strand comprises or consists of a modifiedsequence of any one of the modified sequences in Table 4.

In some embodiments, the sense and antisense strands of the RNAi agentsdescribed herein contain the same number of nucleotides. In someembodiments, the sense and antisense strands of the RNAi agentsdescribed herein contain different numbers of nucleotides. In someembodiments, the sense strand 5′ end and the antisense strand 3′ end ofan RNAi agent form a blunt end. In some embodiments, the sense strand 3′end and the antisense strand 5′ end of an RNAi agent form a blunt end.In some embodiments, both ends of an RNAi agent form blunt ends. In someembodiments, neither end of an RNAi agent is blunt-ended. As used hereina “blunt end” refers to an end of a double stranded RNAi agent in whichthe terminal nucleotides of the two annealed strands are complementary(form a complementary base-pair).

In some embodiments, the sense strand 5′ end and the antisense strand 3′end of an RNAi agent form a frayed end. In some embodiments, the sensestrand 3′ end and the antisense strand 5′ end of an RNAi agent form afrayed end. In some embodiments, both ends of an RNAi agent form afrayed end. In some embodiments, neither end of an RNAi agent is afrayed end. As used herein, a frayed end refers to an end of a doublestranded RNAi agent in which the terminal nucleotides of the twoannealed strands from a pair (i.e., do not form an overhang) but are notcomplementary (i.e. form a non-complementary pair). In some embodiments,one or more unpaired nucleotides at the end of one strand of a doublestranded RNAi agent form an overhang. The unpaired nucleotides may be onthe sense strand or the antisense strand, creating either 3′ or 5′overhangs. In some embodiments, the RNAi agent contains: a blunt end anda frayed end, a blunt end and 5′ overhang end, a blunt end and a 3′overhang end, a frayed end and a 5′ overhang end, a frayed end and a 3′overhang end, two 5′ overhang ends, two 3′ overhang ends, a 5′ overhangend and a 3′ overhang end, two frayed ends, or two blunt ends.Typically, when present, overhangs are located at the 3′ terminal endsof the sense strand, the antisense strand, or both the sense strand andthe antisense strand.

Modified nucleotides, when used in various polynucleotide oroligonucleotide constructs, can preserve activity of the compound incells while at the same time increasing the serum stability of thesecompounds, and can also minimize the possibility of activatinginterferon activity in humans upon administering of the polynucleotideor oligonucleotide construct.

In some embodiments, an alpha-ENaC RNAi agent is prepared or provided asa salt, mixed salt, or a free-acid. In some embodiments, an alpha-ENaCRNAi agent is prepared as a sodium salt. Such forms that are well knownin the art are within the scope of the inventions disclosed herein.

Modified Nucleotides

In some embodiments, an alpha-ENaC RNAi agent contains one or moremodified nucleotides. As used herein, a “modified nucleotide” is anucleotide other than a ribonucleotide (2′-hydroxyl nucleotide). In someembodiments, at least 50% (e.g., at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 97%, at least 98%, at least99%, or 100%) of the nucleotides are modified nucleotides. As usedherein, modified nucleotides can include, but are not limited to,deoxyribonucleotides, nucleotide mimics, abasic nucleotides (representedherein as Ab), 2′-modified nucleotides, 3′ to 3′ linkages (inverted)nucleotides (represented herein as invdN, invN, invn), modifiednucleobase-comprising nucleotides, bridged nucleotides, peptide nucleicacids (PNAs), 2′,3′-seco nucleotide mimics (unlocked nucleobaseanalogues, represented herein as N_(UNA) or NUNA), locked nucleotides(represented herein as N_(LNA) or NLNA), 3′-O-methoxy (2′internucleoside linked) nucleotides (represented herein as 3′-OMen),2′-F-Arabino nucleotides (represented herein as NfANA or Nf_(ANA)),5′-Me, 2′-fluoro nucleotide (represented herein as 5Me-Nf), morpholinonucleotides, vinyl phosphonate deoxyribonucleotides (represented hereinas vpdN), vinyl phosphonate containing nucleotides, and cyclopropylphosphonate containing nucleotides (cPrpN). 2′-modified nucleotides(i.e., a nucleotide with a group other than a hydroxyl group at the 2′position of the five-membered sugar ring) include, but are not limitedto, 2′-O-methyl nucleotides (represented herein as a lower case letter‘n’ in a nucleotide sequence), 2′-deoxy-2′-fluoro nucleotides (alsoreferred to herein as 2′-fluoro nucleotide, and represented herein asNf), 2′-deoxy nucleotides (represented herein as dN), 2′-methoxyethyl(2′-O-2-methoxylethyl) nucleotides (also referred to herein as 2′-MOE,and represented herein as NM), 2′-amino nucleotides, and 2′-alkylnucleotides. It is not necessary for all positions in a given compoundto be uniformly modified. Conversely, more than one modification can beincorporated in a single alpha-ENaC RNAi agent or even in a singlenucleotide thereof. The alpha-ENaC RNAi agent sense strands andantisense strands can be synthesized and/or modified by methods known inthe art. Modification at one nucleotide is independent of modificationat another nucleotide.

Modified nucleobases include synthetic and natural nucleobases, such as5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl(e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives ofadenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or2-n-butyl) and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adeninesand guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine,7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

In some embodiments, all or substantially all of the nucleotides of anRNAi agent are modified nucleotides. As used herein, an RNAi agentwherein substantially all of the nucleotides present are modifiednucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or4) nucleotides in both the sense strand and the antisense strand beingribonucleotides (i.e., unmodified). As used herein, a sense strandwherein substantially all of the nucleotides present are modifiednucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2)nucleotides in the sense strand being unmodified ribonucleotides. Asused herein, an antisense sense strand wherein substantially all of thenucleotides present are modified nucleotides is an antisense strandhaving two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strandbeing unmodified ribonucleotides. In some embodiments, one or morenucleotides of an RNAi agent is an unmodified ribonucleotide.

Modified Internucleoside Linkages

In some embodiments, one or more nucleotides of an alpha-ENaC RNAi agentare linked by non-standard linkages or backbones (i.e., modifiedinternucleoside linkages or modified backbones). Modifiedinternucleoside linkages or backbones include, but are not limited to,phosphorothioate groups (represented herein as a lower case “s”), chiralphosphorothioates, thiophosphates, phosphorodithioates,phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g.,methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates,phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate,aminoalkylphosphoramidates, or thionophosphoramidates),thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholinolinkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linkedanalogs of boranophosphates, or boranophosphates having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. In some embodiments, a modifiedinternucleoside linkage or backbone lacks a phosphorus atom. Modifiedinternucleoside linkages lacking a phosphorus atom include, but are notlimited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixedheteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or moreshort chain heteroatomic or heterocyclic inter-sugar linkages. In someembodiments, modified internucleoside backbones include, but are notlimited to, siloxane backbones, sulfide backbones, sulfoxide backbones,sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones,sulfamate backbones, methyleneimino and methylenehydrazino backbones,sulfonate and sulfonamide backbones, amide backbones, and otherbackbones having mixed N, O, S, and CH₂ components.

In some embodiments, a sense strand of an alpha-ENaC RNAi agent cancontain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisensestrand of an alpha-ENaC RNAi agent can contain 1, 2, 3, 4, 5, or 6phosphorothioate linkages, or both the sense strand and the antisensestrand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioatelinkages. In some embodiments, a sense strand of an alpha-ENaC RNAiagent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisensestrand of an alpha-ENaC RNAi agent can contain 1, 2, 3, or 4phosphorothioate linkages, or both the sense strand and the antisensestrand independently can contain 1, 2, 3, or 4 phosphorothioatelinkages.

In some embodiments, an alpha-ENaC RNAi agent sense strand contains atleast two phosphorothioate internucleoside linkages. In someembodiments, the at least two phosphorothioate internucleoside linkagesare between the nucleotides at positions 1-3 from the 3′ end of thesense strand. In some embodiments, one phosphorothioate internucleosidelinkage is at the 5′ end of the sense strand, and anotherphosphorothioate linkage is at the 3′ end of the sense strand. In someembodiments, the at least two phosphorothioate internucleoside linkagesare between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8from the 5′ end of the sense strand. In some embodiments, an alpha-ENaCRNAi agent antisense strand contains four phosphorothioateinternucleoside linkages. In some embodiments, the four phosphorothioateinternucleoside linkages are between the nucleotides at positions 1-3from the 5′ end of the antisense strand and between the nucleotides atpositions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5′ end.In some embodiments, an alpha-ENaC RNAi agent contains at least twophosphorothioate internucleoside linkages in the sense strand and threeor four phosphorothioate internucleoside linkages in the antisensestrand.

In some embodiments, an alpha-ENaC RNAi agent contains one or moremodified nucleotides and one or more modified internucleoside linkages.In some embodiments, a 2′-modified nucleoside is combined with modifiedinternucleoside linkage.

Alpha-ENaC RNAi Agents

In some embodiments, the alpha-ENaC RNAi agents disclosed herein targetan alpha-ENaC gene at or near the positions of the alpha-ENaC sequenceshown in Table 1. In some embodiments, the antisense strand of analpha-ENaC RNAi agent disclosed herein includes a core stretch sequencethat is fully, substantially, or at least partially complementary to atarget alpha-ENaC 19-mer sequence disclosed in Table 1.

TABLE 1  Alpha-ENaC 19-mer mRNA Target Sequences (taken from homo sapiens sodium channel epithelial 1 alpha subunit (SCNN1A), transcript variant 1, GenBank NM_001038.5 (SEQ ID NO: 1)) alpha-ENaC 19-mer CorrespondingTarget Sequences Positions on  SEQ ID No. (5′→3′) SEQ ID NO: 1 11UGUGCAACCAGAACAAAUC  972-990 12 GUGCAACCAGAACAAAUCG  973-991 13GCAGAGCAGAAUGACUUCA 1289-1307 14 AGAGCAGAAUGACUUCAUU 1291-1309 15CUACCAGACAUACUCAUCA 1000-1018 16 UCUACCAGACAUACUCAUC  999-1017 17CUUUGACCUGUACAAAUAC  763-781 18 UGGAAGGACUGGAAGAUCG  944-962 19GGAAGGACUGGAAGAUCGG  945-963 20 CUGUGCCUACAUCUUCUAU 1579-1597

In some embodiments, an alpha-ENaC RNAi agent includes an antisensestrand wherein position 19 of the antisense strand (5′→3′) is capable offorming a base pair with position 1 of a 19-mer target sequencedisclosed in Table 1. In some embodiments, an alpha-ENaC agent includesan antisense strand wherein position 1 of the antisense strand (5′→3′)is capable of forming a base pair with position 19 of a 19-mer targetsequence disclosed in Table 1.

In some embodiments, an alpha-ENaC agent includes an antisense strandwherein position 2 of the antisense strand (5′→3′) is capable of forminga base pair with position 18 of a 19-mer target sequence disclosed inTable 1. In some embodiments, an alpha-ENaC agent includes an antisensestrand wherein positions 2 through 18 of the antisense strand (5′→3′)are capable of forming base pairs with each of the respectivecomplementary bases located at positions 18 through 2 of the 19-mertarget sequence disclosed in Table 1.

For the RNAi agents disclosed herein, the nucleotide at position 1 ofthe antisense strand (from 5′ end→3′ end) can be perfectly complementaryto the alpha-ENaC gene, or can be non-complementary to the alpha-ENaCgene. In some embodiments, the nucleotide at position 1 of the antisensestrand (from 5′ end→3′ end) is a U, A, or dT. In some embodiments, thenucleotide at position 1 of the antisense strand (from 5′ end→3′ end)forms an A:U or U:A base pair with the sense strand.

In some embodiments, an alpha-ENaC RNAi agent antisense strand comprisesthe sequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any ofthe antisense strand sequences in Table 2 or Table 3. In someembodiments, an alpha-ENaC RNAi sense strand comprises the sequence ofnucleotides (from 5′ end→3′ end) 1-17, 1-18, or 2-18 of any of the sensestrand sequences in Table 2 or Table 4.

In some embodiments, an alpha-ENaC RNAi agent is comprised of (i) anantisense strand comprising the sequence of nucleotides (from 5′ end→3′end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2 orTable 3, and (ii) a sense strand comprising the sequence of nucleotides(from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strand sequencesin Table 2 or Table 4.

In some embodiments, the alpha-ENaC RNAi agents include core 19-mernucleotide sequences shown in the following Table 2.

TABLE 2  Alpha-ENaC RNAi Agent Antisense Strand and  Sense Strand Core Stretch Base Sequences (N = any nucleobase)  Antisense Strand Base   Sense Strand Base SEQ Sequence (5′→3′) SEQSequence (5′→3′) Corresponding ID (Shown as an Unmodified ID(Shown as an Unmodified Positions on NO: Nucleotide Sequence) NO:Nucleotide Sequence) SEQ ID NO: 1 21 UAUUUGUUCUGGUUGCACA 60UGUGCAACCAGAACAAAUA  972-990 22 AAUUUGUUCUGGUUGCACA 61UGUGCAACCAGAACAAAUU  972-990 23 GAUUUGUUCUGGUUGCACA 62UGUGCAACCAGAACAAAUC  972-990 24 NAUUUGUUCUGGUUGCACA 63UGUGCAACCAGAACAAAUN  972-990 25 NAUUUGUUCUGGUUGCACN 64NGUGCAACCAGAACAAAUN  972-990 26 AAUGAAGUCAUUCUGCUCU 65AGAGCAGAAUGACUUCAUU 1291-1309 27 UAUGAAGUCAUUCUGCUCU 66AGAGCAGAAUGACUUCAUA 1291-1309 28 NAUGAAGUCAUUCUGCUCU 67AGAGCAGAAUGACUUCAUN 1291-1309 29 NAUGAAGUCAUUCUGCUCN 68NGAGCAGAAUGACUUCAUN 1291-1309 30 UGAUGAGUAUGUCUGGUAG 69CUACCAGACAUACUCAUCA 1000-1018 31 NGAUGAGUAUGUCUGGUAG 70CUACCAGACAUACUCAUCN 1000-1018 32 NGAUGAGUAUGUCUGGUAN 71NUACCAGACAUACUCAUCN 1000-1018 33 GAUGAGUAUGUCUGGUAGA 72UCUACCAGACAUACUCAUC  999-1017 34 UAUGAGUAUGUCUGGUAGA 73UCUACCAGACAUACUCAUA  999-1017 35 NAUGAGUAUGUCUGGUAGA 74UCUACCAGACAUACUCAUN  999-1017 36 NAUGAGUAUGUCUGGUAGN 75NCUACCAGACAUACUCAUN  999-1017 37 CGAUUUGUUCUGGUUGCAC 76GUGCAACCAGAACAAAUCG  973-991 38 UGAUUUGUUCUGGUUGCAC 77GUGCAACCAGAACAAAUCA  973-991 39 NGAUUUGUUCUGGUUGCAC 78GUGCAACCAGAACAAAUCN  973-991 40 NGAUUUGUUCUGGUUGCAN 79NUGCAACCAGAACAAAUCN  973-991 41 GUAUUUGUACAGGUCAAAG 80CUUUGACCUGUACAAAUAC  763-781 42 UUAUUUGUACAGGUCAAAG 81CUUUGACCUGUACAAAUAA  763-781 43 NUAUUUGUACAGGUCAAAG 82CUUUGACCUGUACAAAUAN  763-781 44 NUAUUUGUACAGGUCAAAN 83NUUUGACCUGUACAAAUAN  763-781 45 CGAUCUUCCAGUCCUUCCA 84UGGAAGGACUGGAAGAUCG  944-962 46 UGAUCUUCCAGUCCUUCCA 85UGGAAGGACUGGAAGAUCA  944-962 47 NGAUCUUCCAGUCCUUCCA 86UGGAAGGACUGGAAGAUCN  944-962 48 NGAUCUUCCAGUCCUUCCN 87NGGAAGGACUGGAAGAUCN  944-962 49 CCGAUCUUCCAGUCCUUCC 88GGAAGGACUGGAAGAUCGG  945-963 50 UCGAUCUUCCAGUCCUUCC 89GGAAGGACUGGAAGAUCGA  945-963 51 NCGAUCUUCCAGUCCUUCC 90GGAAGGACUGGAAGAUCGN  945-963 52 NCGAUCUUCCAGUCCUUCN 91NGAAGGACUGGAAGAUCGN  945-963 53 UGAAGUCAUUCUGCUCUGC 92GCAGAGCAGAAUGACUUCA 1289-1307 54 NGAAGUCAUUCUGCUCUGC 93GCAGAGCAGAAUGACUUCN 1289-1307 55 NGAAGUCAUUCUGCUCUGN 94NCAGAGCAGAAUGACUUCN 1289-1307 56 AUAGAAGAUGUAGGCACAG 95CUGUGCCUACAUCUUCUAU 1579-1597 57 UUAGAAGAUGUAGGCACAG 96CUGUGCCUACAUCUUCUAA 1579-1597 58 NUAGAAGAUGUAGGCACAG 97CUGUGCCUACAUCUUCUAN 1579-1597 59 NUAGAAGAUGUAGGCACAN 98NUGUGCCUACAUCUUCUAN 1579-1597

The alpha-ENaC RNAi agent sense strands and antisense strands thatcomprise or consist of the nucleotide sequences in Table 2 can bemodified nucleotides or unmodified nucleotides. In some embodiments, thealpha-ENaC RNAi agents having the sense and antisense strand sequencesthat comprise or consist of any of the nucleotide sequences in Table 2are all or substantially all modified nucleotides.

In some embodiments, the antisense strand of an alpha-ENaC RNAi agentdisclosed herein differs by 0, 1, 2, or 3 nucleotides from any of theantisense strand sequences in Table 2. In some embodiments, the sensestrand of an alpha-ENaC RNAi agent disclosed herein differs by 0, 1, 2,or 3 nucleotides from any of the sense strand sequences in Table 2.

As used herein, each N listed in a sequence disclosed in Table 2 may beindependently selected from any and all nucleobases (including thosefound on both modified and unmodified nucleotides). In some embodiments,an N nucleotide listed in a sequence disclosed in Table 2 has anucleobase that is complementary to the N nucleotide at thecorresponding position on the other strand. In some embodiments, an Nnucleotide listed in a sequence disclosed in Table 2 has a nucleobasethat is not complementary to the N nucleotide at the correspondingposition on the other strand. In some embodiments, an N nucleotidelisted in a sequence disclosed in Table 2 has a nucleobase that is thesame as the N nucleotide at the corresponding position on the otherstrand. In some embodiments, an N nucleotide listed in a sequencedisclosed in Table 2 has a nucleobase that is different from the Nnucleotide at the corresponding position on the other strand.

Certain modified alpha-ENaC RNAi agent sense and antisense strands areprovided in Table 3 and Table 4. Modified alpha-ENaC RNAi agentantisense strands, as well as their underlying unmodified nucleobasesequences, are provided in Table 3. Modified alpha-ENaC RNAi agent sensestrands, as well as their underlying unmodified nucleobase sequences,are provided in Table 4. In forming alpha-ENaC RNAi agents, each of thenucleotides in each of the underlying base sequences listed in Tables 3and 4, as well as in Table 2, above, can be a modified nucleotide.

The alpha-ENaC RNAi agents described herein are formed by annealing anantisense strand with a sense strand. A sense strand containing asequence listed in Table 2, or Table 4 can be hybridized to anyantisense strand containing a sequence listed in Table 2 or Table 3,provided the two sequences have a region of at least 85% complementarityover a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.

In some embodiments, an alpha-ENaC RNAi agent antisense strand comprisesa nucleotide sequence of any of the sequences in Table 2 or Table 3.

In some embodiments, an alpha-ENaC RNAi agent comprises or consists of aduplex having the nucleobase sequences of the sense strand and theantisense strand of any of the sequences in Table 2, Table 3, or Table4.

Examples of antisense strands containing modified nucleotides areprovided in Table 3. Examples of sense strands containing modifiednucleotides are provided in Table 4.

As used in Tables 3 and 4, the following notations are used to indicatemodified nucleotides, targeting groups, and linking groups:

-   -   A=adenosine-3′-phosphate    -   C=cytidine-3′-phosphate    -   G=guanosine-3′-phosphate    -   U=uridine-3′-phosphate    -   I=inosine-3′-phosphate    -   a=2′-O-methyladenosine-3′-phosphate    -   as=2′-O-methyladenosine-3′-phosphorothioate    -   c=2′-O-methylcytidine-3′-phosphate    -   cs=2′-O-methylcytidine-3′-phosphorothioate    -   g=2′-O-methylguanosine-3′-phosphate    -   gs=2′-O-methylguanosine-3′-phosphorothioate    -   i=2′-O-methylinosine-3′-phosphate    -   iS=2′-O-methylinosine-3′-phosphorothioate    -   t=2′-O-methyl-5-methyluridine-3′-phosphate    -   ts=2′-O-methyl-5-methyluridine-3′-phosphorothioate    -   u=2′-O-methyluridine-3′-phosphate    -   us=2′-O-methyluridine-3′-phosphorothioate    -   Nf=any 2′-fluoro modified nucleotide    -   Af=2′-fluoroadenosine-3′-phosphate    -   Afs=2′-fluoroadenosine-3′-phosporothioate    -   Cf=2′-fluorocytidine-3′-phosphate    -   Cfs=2′-fluorocytidine-3′-phosphorothioate    -   Gf=2′-fluoroguanosine-3′-phosphate    -   Gfs=2′-fluoroguanosine-3′-phosphorothioate    -   Tf=2′-fluoro-5′-methyluridine-3′-phosphate    -   Tfs=2′-fluoro-5′-methyluridine-3′-phosphorothioate    -   Uf=2′-fluorouridine-3′-phosphate    -   Ufs=2′-fluorouridine-3′-phosphorothioate    -   dN=any 2′-deoxyribonucleotide    -   dT=2′-deoxythymidine-3′-phosphate    -   N_(UNA)=2′,3′-seco nucleotide mimics (unlocked nucleobase        analogs)-3′-Phosphate    -   N_(UNAS)=2′,3′-seco nucleotide mimics (unlocked nucleobase        analogs)-3′-Phosphorothioate    -   A_(UNA)=2′,3′-seco-adenosine-3′-phosphate    -   A_(UNAS)=2′,3′-seco-adenosine-3′-phosphorothioate    -   C_(UNA)=2′,3′-seco-cytidine-3′-phosphate    -   C_(UNAS)=2′,3′-seco-cytidine-3′-phosphorothioate    -   G_(UNA)=2′,3′-seco-guanosine-3′-phosphate    -   G_(UNAS)=2′,3′-seco-guanosine-3′-phosphorothioate    -   U_(UNA)=2′,3′-seco-uridine-3′-phosphate    -   U_(UNAS)=2′,3′-seco-uridine-3′-phosphorothioate    -   a_2N=see Table 7    -   a_2Ns=see Table 7    -   pu_2N=see Table 7    -   pu_2Ns=see Table 7    -   D2us=see Table 7    -   Npu=see Table 7    -   Nus=see Table 7    -   N_(LNA)=locked nucleotide    -   Nf_(ANA)=2′-F-Arabino nucleotide    -   NM=2′-O-(2-methoxyethyl) nucleotide    -   AM=2′-O-(2-methoxyethyl)adenosine-3′-phosphate    -   AMs=2′-O-(2-methoxyethyl)adenosine-3′-phosphorothioate    -   TM=2′-O-(2-methoxyethyl)thymidine-3′-phosphate    -   TMs=2′-O-(2-methoxyethyl)thymidine-3′-phosphorothioate    -   R=ribitol    -   (invdN)=any inverted deoxyribonucleotide (3′-3′ linked        nucleotide)    -   (invAb)=inverted (3′-3′ linked) abasic        deoxyribonucleotide-5′-phosphate, see Table 7    -   (invAb)s=inverted (3′-3′ linked) abasic        deoxyribonucleotide-5′-phosphorothioate, see Table 7    -   (invn)=any inverted 2′-OMe nucleotide (3′-3′ linked nucleotide)    -   s=phosphorothioate linkage    -   vpdN=vinyl phosphonate deoxyribonucleotide    -   (5Me-Nf)=5′-Me, 2′-fluoro nucleotide    -   cPrp=cyclopropyl phosphonate, see Table 7    -   epTcPr=see Table 7    -   epTM=see Table 7    -   spus=see Table 7    -   (Chol-TEG)=see Table 7    -   (TEG-Biotin)=see Table 7    -   (PEG-C3-SS)=see Table 7    -   (Alk-SS-C6)=see Table 7    -   (C6-SS-Alk)=see Table 7    -   (C6-SS-C6)=see Table 7    -   (6-SS-6)=see Table 7    -   (C6-SS-Alk-Me)=see Table 7    -   (NH2-C6)=see Table 7    -   (TriAlk #)=see Table 7    -   (TriAlk #)s=see Table 7

As the person of ordinary skill in the art would readily understand,unless otherwise indicated by the sequence (such as, for example, by aphosphorothioate linkage “s”), when present in an oligonucleotide, thenucleotide monomers are mutually linked by 5′-3′-phosphodiester bonds.Further, the person of ordinary skill in the art would readilyunderstand that the terminal nucleotide at the 3′ end of a givenoligonucleotide sequence would typically have a hydroxyl (—OH) group atthe respective 3′ position of the given monomer instead of a phosphatemoiety ex vivo. Moreover, as the person of ordinary skill would readilyunderstand and appreciate, while the phosphorothioate chemicalstructures depicted herein typically show the anion on the sulfur atom,the inventions disclosed herein encompass all phosphorothioate tautomersand/or diastereomers (e.g., where the sulfur atom has a double-bond andthe anion is on an oxygen atom). Unless expressly indicated otherwiseherein, such understandings of the person of ordinary skill in the artare used when describing the alpha-ENaC RNAi agents and compositions ofalpha-ENaC RNAi agents disclosed herein.

Certain examples of targeting groups and linking groups used with thealpha-ENaC RNAi agents disclosed herein are included in the chemicalstructures provided below in Table 6. Each sense strand and/or antisensestrand can have any targeting groups or linking groups listed herein, aswell as other targeting or linking groups, conjugated to the 5′ and/or3′ end of the sequence.

TABLE 3  Alpha-ENaC RNAi Agent Antisense Strand SequencesUnderlying Base  SEQ Sequence (5′→3′)(Shown  SEQ AS Strand  IDas an Unmodified  ID ID Modified Antisense Strand (5′→3′) NO.Nucleotide Sequence) NO. AM04730-AS usAfsusuuGfuUfcUfgGfuUfgCfaCfaGfcusg 99 UAUUUGUUCUGGUUGCACAGCUG 224 AM05080-ASusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfcusg 100 UAUUUGUUCUGGUUGCACAGCUG 224AM05081-AS usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc   6 UAUUUGUUCUGGUUGCACAGC  7 AM05082-AS usAfsusUfuGfuUfcUfgGfuUfgCfaCfagsc 101UAUUUGUUCUGGUUGCACAGC   7 AM05083-AS usAfsusUfuGfuUfcUfgGfuUfgCfaCfausu102 UAUUUGUUCUGGUUGCACAUU 226 AM05084-ASvpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc 103 UAUUUGUUCUGGUUGCACAGC   7AM05085-AS asAfsusUfuGfuUfcUfgGfuUfgCfaCfagsc 104 AAUUUGUUCUGGUUGCACAGC227 AM05772-AS usAfsusGfaAfgUfcAfuUfcUfgCfuCfuGfsc 105UAUGAAGUCAUUCUGCUCUGC 228 AM05773-AS usGfsasUfgAfgUfaUfgUfcUfgGfuAfgAfsa106 UGAUGAGUAUGUCUGGUAGAA 229 AM05774-ASusGfsasUfuUfgUfuCfuGfgUfuGfcAfcAfsg 107 UGAUUUGUUCUGGUUGCACAG 230AM05775-AS usAfsusGfaGfuAfuGfuCfuGfgUfaGfaAfsg 108 UAUGAGUAUGUCUGGUAGAAG231 AM05776-AS usUfsasUfuUfgUfaCfaGfgUfcAfaAfgAfsg 109UUAUUUGUACAGGUCAAAGAG 232 AM05777-ASusAfsusGfaAfgUfCfAfuUfcUfgCfuCfuGfsc 110 UAUGAAGUCAUUCUGCUCUGC 228AM05778-AS usGfsasUfgAfgUfAfUfgUfcUfgGfuAfgAfsa 111UGAUGAGUAUGUCUGGUAGAA 229 AM05779-ASusGfsasUfuUfgUfUfCfuGfgUfuGfcAfcAfsg 112 UGAUUUGUUCUGGUUGCACAG 230AM05780-AS usAfsusGfaGfuAfUfGfuCfuGfgUfaGfaAfsg 113UAUGAGUAUGUCUGGUAGAAG 231 AM05781-ASusUfsasUfuUfgUfAfCfaGfgUfcAfaAfgAfsg 114 UUAUUUGUACAGGUCAAAGAG 232AM05782-AS usAfsusGfaAfgUfCfAfuUfcUfgCfuCfuusu 115 UAUGAAGUCAUUCUGCUCUUU233 AM05783-AS usGfsasUfgAfgUfAfUfgUfcUfgGfuAfgusu 116UGAUGAGUAUGUCUGGUAGUU 234 AM05784-AS usGfsasUfuUfgUfUfCfuGfgUfuGfcAfcusu117 UGAUUUGUUCUGGUUGCACUU 235 AM05785-ASusAfsusGfaGfuAfUfGfuCfuGfgUfaGfausu 118 UAUGAGUAUGUCUGGUAGAUU 236AM05786-AS usUfsasUfuUfgUfAfCfaGfgUfcAfaAfgusu 119 UUAUUUGUACAGGUCAAAGUU237 AM05916-AS cPrpusAfuUfuGfuUfcUfgGfuUfgCfaCfaGfsc 120UAUUUGUUCUGGUUGCACAGC   7 AM05917-AScPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc 121 UAUUUGUUCUGGUUGCACAGC   7AM06240-AS cPrpuAfuUfuGfuUfcUfgGfuUfgCfaCfaGfc 122 UAUUUGUUCUGGUUGCACAGC  7 AM06460-AS cPrpuAfuUfuGfuUfcUfgGfuUfgCfaCfaGfc(invAb) 123UAUUUGUUCUGGUUGCACAGC   7 AM06461-AScPrpuAfuUfuGfuUfcUfgGfuUfgCfaCfaGfsc 124 UAUUUGUUCUGGUUGCACAGC   7AM06462-AS cPrpusAfsuUfuGfuUfcUfgGfuUfgCfaCfaGfsc 125UAUUUGUUCUGGUUGCACAGC   7 AM06691-AS usGfsasUfcUfuCfcAfgUfcCfuUfcCfaGfsu126 UGAUCUUCCAGUCCUUCCAGU 238 AM06693-ASusCfsgsAfuCfuUfcCfaGfuCfcUfuCfcAfsg 127 UCGAUCUUCCAGUCCUUCCAG 239AM06695-AS usGfsasAfgUfcAfuUfcUfgCfuCfuGfcGfsc 128 UGAAGUCAUUCUGCUCUGCGC240 AM06697-AS asUfsasGfaAfgAfuGfuAfgGfcAfcAfgCfsc 129AUAGAAGAUGUAGGCACAGCC 241 AM06699-AS usAfsusCfgUfgAfcAfgAfgGfgAfgAfcUfsc130 UAUCGUGACAGAGGGAGACUC 242 AM06701-ASusUfsgsAfcCfaUfcGfuGfaCfaGfaGfgGfsa 131 UUGACCAUCGUGACAGAGGGA 243AM06765-AS cPrpuAfuUfuGfuUfcUfgGfuUfgCfaCfaG_(UNA)C_(UNA) 132UAUUUGUUCUGGUUGCACAGC   7 AM06766-AScPrpuAfuUfuGfuUfcUfgGfuUfgCfaCfaGfC_(UNA)U_(UNA) 133UAUUUGUUCUGGUUGCACAGCU 244 AM06767-AScPrpuAfuUfuGfuUfcUfgGfuUfgCfaCfaGfcU_(UNA)U_(UNA) 134UAUUUGUUCUGGUUGCACAGCUU 245 AM07066-AScPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg  10 UAUUUGUUCUGGUUGCACAGG   3AM07170-AS cPrpusAfsusUfuGfU_(UNA)UfcUfgGfuUfgCfaCfaGfsg 135UAUUUGUUCUGGUUGCACAGG   3 AM07174-AScPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsu 136 UAUUUGUUCUGGUUGCACAGU 247AM07200-AS usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg   2 UAUUUGUUCUGGUUGCACAGG  3 AM07204-AS usAfsusUfuGfU_(UNA)UfcUfgGfuUfgCfaCfaGfsg 137UAUUUGUUCUGGUUGCACAGG   3 AM07206-AS usAfsusUfuGfuUfcUfgGfuUfgCfaCfgGfsg138 UAUUUGUUCUGGUUGCACGGG 248 AM07208-ASusAfsusUfuGfuUfcUfgGfuUfgCfaCfgGfsu 139 UAUUUGUUCUGGUUGCACGGU 249AM07333-AS usAfsusUfuGfuUfcUfgGfuUfgCfaCfcGfsu 140 UAUUUGUUCUGGUUGCACCGU250 AM07335-AS usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsu 141UAUUUGUUCUGGUUGCACAGU 247 AM07340-AS usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsa142 UAUUUGUUCUGGUUGCACAGA 251 AM07409-ASpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg 143 UAUUUGUUCUGGUUGCACAGG   3AM07410-AS D2usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg 144UAUUUGUUCUGGUUGCACAGG   3 AM07411-ASspusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg 145 UAUUUGUUCUGGUUGCACAGG   3AM07412-AS epusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg 146UAUUUGUUCUGGUUGCACAGG   3 AM07484-ASU_(UNA)sAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg 147 UAUUUGUUCUGGUUGCACAGG   3AM07485-AS isAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg 148 IAUUUGUUCUGGUUGCACAGG252 AM07496-AS usAfsusUfuguucugGfuUfgCfaCfaGfsu 149UAUUUGUUCUGGUUGCACAGU 247 AM07497-AS usAfsusUfuguucUfgGfuUfgcaCfaGfsu150 UAUUUGUUCUGGUUGCACAGU 247 AM07605-ASTMsAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc 151 TAUUUGUUCUGGUUGCACAGC 253AM07669-AS asGfsasAfgUfcAfuUfcUfgCfuCfuGfcusu 152 AGAAGUCAUUCUGCUCUGCUU254

TABLE 4 Alpha-ENaC Agent Sense Strand Sequences Underlying Base  SEQSequence (5′→3′) SEQ ID (Shown as an Unmodified ID Strand IDModified Sense Strand (5′→3′) NO. Nucleotide Sequence) NO. AM05073-SSgscugugCfaAfcCfaGfaacaaauas(invAb) 153 GCUGUGCAACCAGAACAAAUA 255AM05074-SS gscugugcaAfCfCfagaacaaauas(invAb) 154 GCUGUGCAACCAGAACAAAUA255 AM05075-SS asaugugcaAfCfCfagaacaaauas(invAb) 295AAUGUGCAACCAGAACAAAUA 296 AM05077-SS gscugugcaAfCfCfagaacaaauus(invAb)155 GCUGUGCAACCAGAACAAAUU 256 AM05487-SS(NH2-C6)sgscugugcaAfCfCfagaacaaauas(invAb) 156 GCUGUGCAACCAGAACAAAUA 255AM05787-SS gscagagcaGfAfAfugacuucauas(invAb) 157 GCAGAGCAGAAUGACUUCAUA257 AM05788-SS usucuaccaGfAfCfauacucaucas(invAb) 158UUCUACCAGACAUACUCAUCA 258 AM05789-SS csugugcaaCfCfAfgaacaaaucas(invAb)159 CUGUGCAACCAGAACAAAUCA 259 AM05790-SScsuucuaccAfGfAfcauacucauas(invAb) 160 CUUCUACCAGACAUACUCAUA 260AM05791-SS csucuuugaCfCfUfguacaaauaas(invAb) 161 CUCUUUGACCUGUACAAAUAA261 AM05792-SS (invAb)AfgAfgCfaGfAfAfuGfaCfuUfcauausu(invAb) 162AGAGCAGAAUGACUUCAUAUU 262 AM05793-SS(invAb)CfuAfcCfaGfAfCfaUfaCfuCfaucausu(invAb) 163 CUACCAGACAUACUCAUCAUU263 AM05794-SS (invAb)GfuGfcAfaCfCfAfgAfaCfaAfaucausu(invAb) 164GUGCAACCAGAACAAAUCAUU 264 AM05795-SS(invAb)UfcUfaCfcAfGfAfcAfuAfcUfcauausu(invAb) 165 UCUACCAGACAUACUCAUAUU265 AM05796-SS (invAb)CfuUfuGfaCfCfUfgUfaCfaAfauaausu(invAb) 166CUUUGACCUGUACAAAUAAUU 266 AM06162-SS(invAb)gcugugcaAfCfCfagaacaaaua(invAb) 167 GCUGUGCAACCAGAACAAAUA 255AM06246-SS gcugugcaAfCfCfagaacaaau(invdA) 168 GCUGUGCAACCAGAACAAAUA 255AM06459-SS gcugugcaAfCfCfagaacaaaua(invAb) 169 GCUGUGCAACCAGAACAAAUA 255AM06690-SS (NH2-C6)sascuggaagGfAfCfuggaagaucas(invAb) 170ACUGGAAGGACUGGAAGAUCA 267 AM06692-SS(NH2-C6)scsuggaaggAfCfUfggaagaucgas(invAb) 171 CUGGAAGGACUGGAAGAUCGA 268AM06694-SS (NH2-C6)sgscgcagagCfAfGfaaugacuucas(invAb) 172GCGCAGAGCAGAAUGACUUCA 269 AM06696-SS(NH2-C6)sgsgcugugcCfUfAfcaucuucuaus(invAb) 173 GGCUGUGCCUACAUCUUCUAU 270AM06698-SS (NH2-C6)sgsagucuccCfUfCfugucacgauas(invAb) 174GAGUCUCCCUCUGUCACGAUA 271 AM06700-SS(NH2-C6)suscccucugUfCfAfcgauggucaas(invAb) 175 UCCCUCUGUCACGAUGGUCAA 272AM07064-SS (NH2-C6)gscugugcaAfCfCfagaacaaauas(invAb) 176GCUGUGCAACCAGAACAAAUA 255 AM07065-SS(NH2-C6)scscugugcaAfCfCfagaacaaauas(invAb) 177 CCUGUGCAACCAGAACAAAUA 273AM07067-SS (NH2-C6)cscugugcaAfCfCfagaacaaauas(invAb) 178CCUGUGCAACCAGAACAAAUA 273 AM07169-SS(NH2-C6)scscugugcaAfCfCfaGaacaaauasinvAb) 179 CCUGUGCAACCAGAACAAAUA 273AM07171-SS (NH2-C6)scscugugcaAfCfCfaiaacaaauas(invAb) 180CCUGUGCAACCAIAACAAAUA 274 AM07172-SS(NH2-C6)scscugugcaAfCfCfagaacaa_2Nauas(invAb) 181CCUGUGCAACCAGAACA(A^(2n))AUA 275 AM07173-SS(NH2-C6)sacugugcaAfCfCfagaacaaauas(invAb) 182 ACUGUGCAACCAGAACAAAUA 276AM07201-SS (NH2-C6)cscugugcaAfCfCfaGaacaaauas(invAb) 183CCUGUGCAACCAGAACAAAUA 273 AM07202-SS(NH2-C6)cscugugcaAfCfCfaiaacaaauas(invAb) 184 CCUGUGCAACCAIAACAAAUA 274AM07203-SS (NH2-C6)cscugugcaAfCfUfagaacaaauas(invAb) 185CCUGUGCAACUAGAACAAAUA 277 AM07205-SS(NH2-C6)csccgugcaAfCfCfagaacaaauas(invAb) 186 CCCGUGCAACCAGAACAAAUA 278AM07207-SS (NH2-C6)asccgugcaAfCfCfagaacaaauas(invAb) 187ACCGUGCAACCAGAACAAAUA 279 AM07217-SS(NH2-C6)cscugugcaAfCfCfagaacaaauas(invAb)s 188 CCUGUGCAACCAGAACAAAUA 273(C6-SS-C6) AM07218-SS (NH2-C6)cscugugcaAfCfCfagaacaaauas(invAb) 189CCUGUGCAACCAGAACAAAUA 273 (C6-SS-C6) AM07276-SS(TriAlk1)sgscugugcaAfCfCfagaacaaauas(invAb) 190 GCUGUGCAACCAGAACAAAUA255 AM07280-SS (NH2-C6)cscugugcaAfCfCfagaacaaauas(invAb)s 191CCUGUGCAACCAGAACAAAUA 273 (6-SS-6) AM07281-SS(NH2-C6)cscugugcaAfCfCfagaacaaauas(invAb) 192 CCUGUGCAACCAGAACAAAUA 273(6-SS-6) AM07329-SS (TriAlk1)cscugugcaAfCfCfagaacaaauas(invAb) 193CCUGUGCAACCAGAACAAAUA 273 AM07330-SS(TriAlk2)cscugugcaAfCfCfagaacaaauas(invAb) 194 CCUGUGCAACCAGAACAAAUA 273AM07331-SS (TriAlk3)cscugugcaAfCfCfagaacaaauas(invAb) 195CCUGUGCAACCAGAACAAAUA 273 AM07332-SS(NH2-C6)ascggugcaAfCfCfagaacaaauas(invAb) 196 ACGGUGCAACCAGAACAAAUA 280AM07334-SS (NH2-C6)ascugugcaAfCfCfagaacaaauas(invAb) 197ACUGUGCAACCAGAACAAAUA 276 AM07336-SS(NH2-C6)ascugugcaAfCfCfagaacaaa_2Nuas(invAb) 198ACUGUGCAACCAGAACAA(A^(2n))UA 281 AM07337-SS(NH2-C6)ascugugcaAfCfCfagaacaa_2Nauas(invAb) 199ACUGUGCAACCAGAACA(A^(2n))AUA 282 AM07338-SS(NH2-C6)ascugugcaAfCfCfagaaca_2Naauas(invAb) 200ACUGUGCAACCAGAAC(A^(2n))AAUA 283 AM07339-SS(NH2-C6)uscugugcaAfCfCfagaacaaauas(invAb) 201 UCUGUGCAACCAGAACAAAUA 284AM07341-SS (NH2-C6)cscugugcaAfCfCfagaacaa_2Nauas(invAb) 202CCUGUGCAACCAGAACA(A^(2n))AUA 285 AM07342-SS(NH2-C6)cscugugcaAfCfCfaGaacaa_2Nauas(invAb) 203CCUGUGCAACCAGAACA(A^(2n))AUA 275 AM07343-SS(NH2-C6)cscugugcaAfCfCfagaacaaa_2Nuas(invAb) 204CCUGUGCAACCAGAACAA(A^(2n))UA 286 AM07344-SS(NH2-C6)cscugugcaAfCfCfagaaca_2Naauas(invAb) 205CCUGUGCAACCAGAAC(A^(2n))AAUA 287 AM07400-SS(TriAlk4)cscugugcaAfCfCfagaacaaauas(invAb) 206 CCUGUGCAACCAGAACAAAUA 273AM07401-SS (TriAlk5)cscugugcaAfCfCfagaacaaauas(invAb) 207CCUGUGCAACCAGAACAAAUA 273 AM07402-SS(TriAlk6)cscugugcaAfCfCfagaacaaauas(invAb) 208 CCUGUGCAACCAGAACAAAUA 273AM07486-SS (NH2-C6)cscugugcaAfCfCfagaacaaaucs(invAb) 209CCUGUGCAACCAGAACAAAUC 288 AM07495-SS(NH2-C6)ascUfgUfgCfaAfCfCfagaacaaauas(invAb) 210 ACUGUGCAACCAGAACAAAUA276 AM07498-SS (NH2-C6)ascUfgUfgCfaAfcCfaGfaacaaauas(invAb) 211ACUGUGCAACCAGAACAAAUA 276   AM07499-SS(NH2-C6)ascUfgUfgCfaAfCfCfagaacaa_2Nauas(invAb) 212ACUGUGCAACCAGAACA(A^(2n))AUA 282 AM07594-SS(TriAlk7)cscugugcaAfCfCfagaacaaauas(invAb) 213 CCUGUGCAACCAGAACAAAUA 273AM07595-SS (TriAlk8)cscugugcaAfCfCfagaacaaauas(invAb) 214CCUGUGCAACCAGAACAAAUA 273 AM07606-SS(NH2-C6)sgscugugcaAfCfCfagaacaaauas(invAb) 215 GCUGUGCAACCAGAACAAAUA 255(C6-SS-C6)(invAb) AM07611-SS (TriAlk9)cscugugcaAfCfCfagaacaaauas(invAb)216 CCUGUGCAACCAGAACAAAUA 273 AM07612-SS(TriAlk10)cscugugcaAfCfCfagaacaaauas(invAb) 217 CCUGUGCAACCAGAACAAAUA273 AM07665-SS (NH2-C6)ascuggaagGfAfCfuggaagaucas(invAb) 218ACUGGAAGGACUGGAAGAUCA 267 AM07666-SS(NH2-C6)scsugugcaaCfCfAfgaacaaaucas(invAb) 219 CUGUGCAACCAGAACAAAUCA 259AM07667-SS (NH2-C6)csugugcaaCfCfAfgaacaaaucas(invAb) 220CUGUGCAACCAGAACAAAUCA 259 AM07668-SS(NH2-C6)sgscagagCfAfGfaaugacuucuuus(invAb) 221 GCAGAGCAGAAUGACUUCUUU 289AM07670-SS (NH2-C6)gscagagCfAfGfaaugacuucuuus(invAb) 222GCAGAGCAGAAUGACUUCUUU 289 AM07807-SS(TriAlk14)cscugugcaAfCfCfagaacaaauas(invAb) 223 CCUGUGCAACCAGAACAAAUA273 (A2N) = 2-aminoadenine nucleotide

The alpha-ENaC RNAi agents disclosed herein are formed by annealing anantisense strand with a sense strand. A sense strand containing asequence listed in Table 2 or Table 4 can be hybridized to any antisensestrand containing a sequence listed in Table 2 or Table 3, provided thetwo sequences have a region of at least 85% complementarity over acontiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.

In some embodiments, the antisense strand of an alpha-ENaC RNAi agentdisclosed herein differs by 0, 1, 2, or 3 nucleotides from any of theantisense strand sequences in Table 3. In some embodiments, the sensestrand of an alpha-ENaC RNAi agent disclosed herein differs by 0, 1, 2,or 3 nucleotides from any of the sense strand sequences in Table 4.

In some embodiments, an alpha-ENaC RNAi agent antisense strand comprisesa nucleotide sequence of any of the sequences in Table 2 or Table 3. Insome embodiments, an alpha-ENaC RNAi agent antisense strand comprisesthe sequence of nucleotides (from 5′ end→3′ end) 1-17, 2-17, 1-18, 2-18,1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, or2-24 of any of the sequences in Table 2 or Table 3. In certainembodiments, an alpha-ENaC RNAi agent antisense strand comprises orconsists of a modified sequence of any one of the modified sequences inTable 3.

In some embodiments, an alpha-ENaC RNAi agent sense strand comprises thenucleotide sequence of any of the sequences in Table 2 or Table 4. Insome embodiments, an alpha-ENaC RNAi agent sense strand comprises thesequence of nucleotides (from 5′ end→3′ end) 1-17, 2-17, 3-17, 4-17,1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20,1-21, 2-21, 3-21, 4-21, 1-22, 2-22, 3-22, 4-22, 1-23, 2-23, 3-23, 4-23,1-24, 2-24, 3-24, or 4-24, of any of the sequences in Table 2 or Table4. In certain embodiments, an alpha-ENaC RNAi agent sense strandcomprises or consists of a modified sequence of any one of the modifiedsequences in Table 3.

For the RNAi agents disclosed herein, the nucleotide at position 1 ofthe antisense strand (from 5′ end→3′ end) can be perfectly complementaryto the alpha-ENaC gene, or can be non-complementary to the alpha-ENaCgene. In some embodiments, the nucleotide at position 1 of the antisensestrand (from 5′ end→3′ end) is a U, A, or dT (or a modified version ofU, A or dT). In some embodiments, the nucleotide at position 1 of theantisense strand (from 5′ end→3′ end) forms an A:U or U:A base pair withthe sense strand.

In some embodiments, an alpha-ENaC RNAi agent antisense strand comprisesthe sequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any ofthe antisense strand sequences in Table 2 or Table 3. In someembodiments, an alpha-ENaC RNAi sense strand comprises the sequence ofnucleotides (from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strandsequences in Table 2 or Table 4.

In some embodiments, an alpha-ENaC RNAi agent includes (i) an antisensestrand comprising the sequence of nucleotides (from 5′ end→3′ end) 2-18or 2-19 of any of the antisense strand sequences in Table 2 or Table 3,and (ii) a sense strand comprising the sequence of nucleotides (from 5′end→3′ end) 1-17 or 1-18 of any of the sense strand sequences in Table 2or Table 4.

A sense strand containing a sequence listed in Table 2 or Table 4 can behybridized to any antisense strand containing a sequence listed in Table2 or Table 3 provided the two sequences have a region of at least 85%complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotidesequence. In some embodiments, the alpha-ENaC RNAi agent has a sensestrand consisting of the modified sequence of any of the modifiedsequences in Table 4, and an antisense strand consisting of the modifiedsequence of any of the modified sequences in Table 3. Certainrepresentative sequence pairings are exemplified by the Duplex ID Nos.shown in Table 5.

In some embodiments, an alpha-ENaC RNAi agent comprises, consists of, orconsists essentially of a duplex represented by any one of the Duplex IDNos. presented herein. In some embodiments, an alpha-ENaC RNAi agentconsists of any of the Duplex ID Nos. presented herein. In someembodiments, an alpha-ENaC RNAi agent comprises the sense strand andantisense strand nucleotide sequences of any of the Duplex ID Nos.presented herein. In some embodiments, an alpha-ENaC RNAi agentcomprises the sense strand and antisense strand nucleotide sequences ofany of the Duplex ID Nos. presented herein and a targeting group,linking group, and/or other non-nucleotide group wherein the targetinggroup, linking group, and/or other non-nucleotide group is covalentlylinked (i.e., conjugated) to the sense strand or the antisense strand.In some embodiments, an alpha-ENaC RNAi agent includes the sense strandand antisense strand modified nucleotide sequences of any of the DuplexID Nos. presented herein. In some embodiments, an alpha-ENaC RNAi agentcomprises the sense strand and antisense strand modified nucleotidesequences of any of the Duplex ID Nos. presented herein and a targetinggroup, linking group, and/or other non-nucleotide group, wherein thetargeting group, linking group, and/or other non-nucleotide group iscovalently linked to the sense strand or the antisense strand.

In some embodiments, an alpha-ENaC RNAi agent comprises an antisensestrand and a sense strand having the nucleotide sequences of any of theantisense strand/sense strand duplexes of Table 2 or Table 5, andfurther comprises a targeting group. In some embodiments, an alpha-ENaCRNAi agent comprises an antisense strand and a sense strand having thenucleotide sequences of any of the antisense strand/sense strandduplexes of Table 2 or Table 5, and further comprises one or more αvβ6integrin targeting ligands.

In some embodiments, an alpha-ENaC RNAi agent comprises an antisensestrand and a sense strand having the nucleotide sequences of any of theantisense strand/sense strand duplexes of Table 2 or Table 5, andfurther comprises a targeting group that is an integrin targetingligand. In some embodiments, an alpha-ENaC RNAi agent comprises anantisense strand and a sense strand having the nucleotide sequences ofany of the antisense strand/sense strand duplexes of Table 2 or Table 5,and further comprises one or more αvβ6 integrin targeting ligands orclusters of αvβ6 integrin targeting ligands (e.g., a tridentate αvβ6integrin targeting ligand).

In some embodiments, an alpha-ENaC RNAi agent comprises an antisensestrand and a sense strand having the modified nucleotide sequences ofany of the antisense strand/sense strand duplexes of Table 5.

In some embodiments, an alpha-ENaC RNAi agent comprises an antisensestrand and a sense strand having the modified nucleotide sequences ofany of the antisense strand/sense strand duplexes of Table 5, andfurther comprises an integrin targeting ligand.

In some embodiments, an alpha-ENaC RNAi agent comprises, consists of, orconsists essentially of any of the duplexes of Table 5.

TABLE 5 Alpha-ENaC RNAi Agent Duplexes with Corresponding Sense andAntisense Strand ID Numbers Duplex ID Antisense Strand ID Sense StrandID AD04019 AM04730-AS AM05073-SS AD04020 AM04730-AS AM05074-SS AD04021AM05080-AS AM05074-SS AD04022 AM05081-AS AM05074-SS AD04023 AM05082-ASAM05074-SS AD04024 AM05083-AS AM05075-SS AD04025 AM05084-AS AM05074-SSAD04026 AM05085-AS AM05077-SS AD04526 AM05772-AS AM05787-SS AD04527AM05773-AS AM05788-SS AD04528 AM05774-AS AM05789-SS AD04529 AM05775-ASAM05790-SS AD04530 AM05776-AS AM05791-SS AD04531 AM05777-AS AM05792-SSAD04532 AM05778-AS AM05793-SS AD04533 AM05779-AS AM05794-SS AD04534AM05780-AS AM05795-SS AD04535 AM05781-AS AM05796-SS AD04536 AM05782-ASAM05792-SS AD04537 AM05783-AS AM05793-SS AD04538 AM05784-AS AM05794-SSAD04539 AM05785-AS AM05795-SS AD04540 AM05786-AS AM05796-SS AD04835AM05917-AS AM05487-SS AD04858 AM05917-AS AM05074-SS AD04859 AM06240-ASAM06162-SS AD04976 AM06460-AS AM06459-SS AD04977 AM06461-AS AM06459-SSAD04978 AM05916-AS AM06459-SS AD04979 AM06462-AS AM06459-SS AD04980AM06462-AS AM06246-SS AD05116 AM06691-AS AM06690-SS AD05117 AM06693-ASAM06692-SS AD05118 AM06695-AS AM06694-SS AD05119 AM06697-AS AM06696-SSAD05120 AM06699-AS AM06698-SS AD05121 AM06701-AS AM06700-SS AD05160AM06240-AS AM06459-SS AD05161 AM06765-AS AM06459-SS AD05162 AM06766-ASAM06459-SS AD05163 AM06767-AS AM06459-SS AD05345 AM05917-AS AM07064-SSAD05346 AM07066-AS AM07065-SS AD05347 AM07066-AS AM07067-SS AD05426AM07066-AS AM07169-SS AD05427 AM07170-AS AM07065-SS AD05428 AM07066-ASAM07171-SS AD05429 AM07066-AS AM07172-SS AD05430 AM07174-AS AM07173-SSAD05453 AM07200-AS AM07067-SS AD05454 AM07200-AS AM07201-SS AD05455AM07200-AS AM07202-SS AD05456 AM07200-AS AM07203-SS AD05457 AM07204-ASAM07067-SS AD05458 AM07206-AS AM07205-SS AD05459 AM07208-AS AM07207-SSAD05471 AM07066-AS AM07217-SS AD05472 AM07066-AS AM07218-SS AD05473AM07200-AS AM07217-SS AD05474 AM07200-AS AM07218-SS AD05515 AM05081-ASAM07276-SS AD05548 AM07200-AS AM07280-SS AD05549 AM07200-AS AM07281-SSAD05558 AM07200-AS AM07329-SS AD05559 AM07200-AS AM07330-SS AD05560AM07200-AS AM07331-SS AD05561 AM07333-AS AM07332-SS AD05562 AM07335-ASAM07334-SS AD05563 AM07335-AS AM07336-SS AD05564 AM07335-AS AM07337-SSAD05565 AM07335-AS AM07338-SS AD05566 AM07340-AS AM07339-SS AD05567AM07200-AS AM07341-SS AD05568 AM07200-AS AM07172-SS AD05569 AM07200-ASAM07342-SS AD05570 AM07200-AS AM07343-SS AD05571 AM07200-AS AM07344-SSAD05611 AM07200-AS AM07400-SS AD05612 AM07200-AS AM07401-SS AD05613AM07200-AS AM07402-SS AD05618 AM07409-AS AM07067-SS AD05619 AM07410-ASAM07067-SS AD05622 AM07411-AS AM07067-SS AD05623 AM07412-AS AM07067-SSAD05625 AM05081-AS AM05487-SS AD05671 AM07484-AS AM07067-SS AD05672AM07485-AS AM07067-SS AD05673 AM07485-AS AM07486-SS AD05683 AM07174-ASAM07334-SS AD05684 AM07335-AS AM07495-SS AD05685 AM07496-AS AM07495-SSAD05686 AM07497-AS AM07334-SS AD05687 AM07496-AS AM07498-SS AD05688AM07174-AS AM07337-SS AD05689 AM07335-AS AM07499-SS AD05690 AM07496-ASAM07499-SS AD05691 AM07497-AS AM07337-SS AD05757 AM07200-AS AM07594-SSAD05758 AM07200-AS AM07595-SS AD05772 AM07605-AS AM05487-SS AD05773AM05081-AS AM07606-SS AD05778 AM07200-AS AM07611-SS AD05779 AM07200-ASAM07612-SS AD05829 AM06691-AS AM07665-SS AD05830 AM05774-AS AM07666-SSAD05831 AM05774-AS AM07667-SS AD05832 AM07669-AS AM07668-SS AD05833AM07669-AS AM07670-SS AD05924 AM07200-AS AM07807-SS

In some embodiments, an alpha-ENaC RNAi agent is prepared or provided asa salt, mixed salt, or a free-acid. The RNAi agents described herein,upon delivery to a cell expressing an alpha-ENaC gene, inhibit orknockdown expression of one or more alpha-ENaC genes in vivo and/or invitro.

Targeting Groups, Linking Groups, Pharmacokinetic (PK) Modulators, andDelivery Vehicles

In some embodiments, an alpha-ENaC RNAi agent contains or is conjugatedto one or more non-nucleotide groups including, but not limited to, atargeting group, a linking group, a pharmacokinetic (PK) modulator, adelivery polymer, or a delivery vehicle. The non-nucleotide group canenhance targeting, delivery, or attachment of the RNAi agent. Examplesof targeting groups and linking groups are provided in Table 6. Thenon-nucleotide group can be covalently linked to the 3′ and/or 5′ end ofeither the sense strand and/or the antisense strand. In someembodiments, an alpha-ENaC RNAi agent contains a non-nucleotide grouplinked to the 3′ and/or 5′ end of the sense strand. In some embodiments,a non-nucleotide group is linked to the 5′ end of an alpha-ENaC RNAiagent sense strand. A non-nucleotide group can be linked directly orindirectly to the RNAi agent via a linker/linking group. In someembodiments, a non-nucleotide group is linked to the RNAi agent via alabile, cleavable, or reversible bond or linker.

In some embodiments, a non-nucleotide group enhances the pharmacokineticor biodistribution properties of an RNAi agent or conjugate to which itis attached to improve cell- or tissue-specific distribution andcell-specific uptake of the conjugate. In some embodiments, anon-nucleotide group enhances endocytosis of the RNAi agent.

Targeting groups or targeting moieties enhance the pharmacokinetic orbiodistribution properties of a conjugate or RNAi agent to which theyare attached to improve cell-specific (including, in some cases, organspecific) distribution and cell-specific (or organ specific) uptake ofthe conjugate or RNAi agent. A targeting group can be monovalent,divalent, trivalent, tetravalent, or have higher valency for the targetto which it is directed. Representative targeting groups include,without limitation, compounds with affinity to cell surface molecule,cell receptor ligands, hapten, antibodies, monoclonal antibodies,antibody fragments, and antibody mimics with affinity to cell surfacemolecules. In some embodiments, a targeting group is linked to an RNAiagent using a linker, such as a PEG linker or one, two, or three abasicand/or ribitol (abasic ribose) residues, which in some instances canserve as linkers. In some embodiments, a targeting group comprises anintegrin targeting ligand.

The alpha-ENaC RNAi agents described herein can be synthesized having areactive group, such as an amino group (also referred to herein as anamine), at the 5′-terminus and/or the 3′-terminus. The reactive groupcan be used subsequently to attach a targeting moiety using methodstypical in the art.

For example, in some embodiments, the alpha-ENaC RNAi agents disclosedherein are synthesized having an NH₂—C₆ group at the 5′-terminus of thesense strand of the RNAi agent. The terminal amino group subsequentlycan be reacted to form a conjugate with, for example, a group thatincludes an αvβ6 integrin targeting ligand. In some embodiments, thealpha-ENaC RNAi agents disclosed herein are synthesized having one ormore alkyne groups at the 5′-terminus of the sense strand of the RNAiagent. The terminal alkyne group(s) can subsequently be reacted to forma conjugate with, for example, a group that includes an αvβ6 integrintargeting ligand.

In some embodiments, a targeting group comprises an integrin targetingligand. In some embodiments, an integrin targeting ligand is an αvβ6integrin targeting ligand. The use of an αvβ6 integrin targeting ligandfacilitates cell-specific targeting to cells having αvβ6 on itsrespective surface, and binding of the integrin targeting ligand canfacilitate entry of the therapeutic agent, such as an RNAi agent, towhich it is linked, into cells such as epithelial cells, includingpulmonary epithelial cells and renal epithelial cells. Integrintargeting ligands can be monomeric or monovalent (e.g., having a singleintegrin targeting moiety) or multimeric or multivalent (e.g., havingmultiple integrin targeting moieties). The targeting group can beattached to the 3′ and/or 5′ end of the RNAi oligonucleotide usingmethods known in the art. The preparation of targeting groups, such asαvβ6 integrin targeting ligands, is described, for example, inInternational Patent Application Publication No. WO 2018/085415 and inU.S. Provisional Patent Application Nos. 62/580,398 and 62/646,739, thecontents of each of which are incorporated herein in its entirety.

Embodiments of the present disclosure include pharmaceuticalcompositions for delivering an alpha-ENaC RNAi agent to a pulmonaryepithelial cell in vivo. Such pharmaceutical compositions can include,for example, an alpha-ENaC RNAi agent conjugated to a targeting groupthat comprises an integrin targeting ligand. In some embodiments, theintegrin targeting ligand is comprised of an αvβ6 integrin ligand.

In some embodiments, a linking group is conjugated to the RNAi agent.The linking group facilitates covalent linkage of the agent to atargeting group, pharmacokinetic modulator, delivery polymer, ordelivery vehicle. The linking group can be linked to the 3′ and/or the5′ end of the RNAi agent sense strand or antisense strand. In someembodiments, the linking group is linked to the RNAi agent sense strand.In some embodiments, the linking group is conjugated to the 5′ or 3′ endof an RNAi agent sense strand. In some embodiments, a linking group isconjugated to the 5′ end of an RNAi agent sense strand. Examples oflinking groups, include, but are not limited to: Alk-SMPT-C6, Alk-SS-C6,DBCO-TEG, Me-Alk-SS-C6, and C6-SS-Alk-Me, reactive groups such a primaryamines and alkynes, alkyl groups, abasic residues/nucleotides, aminoacids, tri-alkyne functionalized groups, ribitol, and/or PEG groups.

A linker or linking group is a connection between two atoms that linksone chemical group (such as an RNAi agent) or segment of interest toanother chemical group (such as a targeting group, pharmacokineticmodulator, or delivery polymer) or segment of interest via one or morecovalent bonds. A labile linkage contains a labile bond. A linkage canoptionally include a spacer that increases the distance between the twojoined atoms. A spacer may further add flexibility and/or length to thelinkage. Spacers include, but are not be limited to, alkyl groups,alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenylgroups, and aralkynyl groups; each of which can contain one or moreheteroatoms, heterocycles, amino acids, nucleotides, and saccharides.Spacer groups are well known in the art and the preceding list is notmeant to limit the scope of the description.

In some embodiments, targeting groups are linked to the alpha-ENaC RNAiagents without the use of an additional linker. In some embodiments, thetargeting group is designed having a linker readily present tofacilitate the linkage to an alpha-ENaC RNAi agent. In some embodiments,when two or more RNAi agents are included in a composition, the two ormore RNAi agents can be linked to their respective targeting groupsusing the same linkers. In some embodiments, when two or more RNAiagents are included in a composition, the two or more RNAi agents arelinked to their respective targeting groups using different linkers.

Any of the alpha-ENaC RNAi agent nucleotide sequences listed in Tables2, 3, and 4, whether modified or unmodified, can contain 3′ and/or 5′targeting group(s), linking group(s), and/or pharmacokineticmodulator(s). Any of the alpha-ENaC RNAi agent sequences listed inTables 3 and 4, or are otherwise described herein, which contain a 3′ or5′ targeting group, linking group, or pharmacokinetic modulator canalternatively contain no 3′ or 5′ targeting group, linking group, orpharmacokinetic modulator, or can contain a different 3′ or 5′ targetinggroup, linking group, or pharmacokinetic modulator including, but notlimited to, those depicted in Table 6. Any of the alpha-ENaC RNAi agentduplexes listed in Table 5, whether modified or unmodified, can furthercomprise a targeting group or linking group, including, but not limitedto, those depicted in Table 6, and the targeting group or linking groupcan be attached to the 3′ or 5′ terminus of either the sense strand orthe antisense strand of the alpha-ENaC RNAi agent duplex.

Examples of certain targeting groups and linking groups are provided inTable 6.

TABLE 6 Structures Representing Various Modified Nucleotides, TargetingGroups, and Linking Groups

Alternatively, other linking groups known in the art may be used.

In some embodiments, a delivery vehicle may be used to deliver an RNAiagent to a cell or tissue. A delivery vehicle is a compound thatimproves delivery of the RNAi agent to a cell or tissue. A deliveryvehicle can include, or consist of, but is not limited to: a polymer,such as an amphipathic polymer, a membrane active polymer, a peptide, amelittin peptide, a melittin-like peptide (MLP), a lipid, a reversiblymodified polymer or peptide, or a reversibly modified membrane activepolyamine.

In some embodiments, the RNAi agents can be combined with lipids,nanoparticles, polymers, liposomes, micelles, DPCs or other deliverysystems available in the art. The RNAi agents can also be chemicallyconjugated to targeting groups, lipids (including, but not limited tocholesterol and cholesteryl derivatives), nanoparticles, polymers,liposomes, micelles, DPCs (see, for example WO 2000/053722, WO2008/022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO2013/158141, each of which is incorporated herein by reference), orother delivery systems available in the art.

Pharmaceutical Compositions and Formulations

The alpha-ENaC RNAi agents disclosed herein can be prepared aspharmaceutical compositions or formulations (also referred to herein as“medicaments”). In some embodiments, pharmaceutical compositions includeat least one alpha-ENaC RNAi agent. These pharmaceutical compositionsare particularly useful in the inhibition of the expression ofalpha-ENaC mRNA in a target cell, a group of cells, a tissue, or anorganism. The pharmaceutical compositions can be used to treat a subjecthaving a disease, disorder, or condition that would benefit fromreduction in the level of the target mRNA, or inhibition in expressionof the target gene. The pharmaceutical compositions can be used to treata subject at risk of developing a disease or disorder that would benefitfrom reduction of the level of the target mRNA or an inhibition inexpression the target gene. In one embodiment, the method includesadministering an alpha-ENaC RNAi agent linked to a targeting ligand asdescribed herein, to a subject to be treated. In some embodiments, oneor more pharmaceutically acceptable excipients (including vehicles,carriers, diluents, and/or delivery polymers) are added to thepharmaceutical compositions that include an alpha-ENaC RNAi agent,thereby forming a pharmaceutical formulation or medicament suitable forin vivo delivery to a subject, including a human.

The pharmaceutical compositions that include an alpha-ENaC RNAi agentand methods disclosed herein decrease the level of the target mRNA in acell, group of cells, group of cells, tissue, organ, or subject,including by administering to the subject a therapeutically effectiveamount of a herein described alpha-ENaC RNAi agent, thereby inhibitingthe expression of alpha-ENaC mRNA in the subject. In some embodiments,the subject has been previously identified or diagnosed as having adisease or disorder that is mediated at least in part by ENaCexpression. In some embodiments, the subject has been previouslyidentified or diagnosed as having enhanced ENaC activity in one or morecells or tissues. In some embodiments, the subject has been previouslydiagnosed with having one or more respiratory diseases such as cysticfibrosis, chronic bronchitis, non-cystic fibrosis bronchiectasis,chronic obstructive pulmonary disease (COPD), asthma, respiratory tractinfections, primary ciliary dyskinesia, and lung carcinoma cysticfibrosis. In some embodiments, the subject has been previously diagnosedwith having one or more ocular diseases such as dry eye. In someembodiments, the subject has been suffering from symptoms associatedwith one or more respiratory diseases that is associated with or causedby enhanced ENaC activity.

In some embodiments, the described pharmaceutical compositions includingan alpha-ENaC RNAi agent are used for treating or managing clinicalpresentations in a subject that would benefit from the inhibition ofexpression of ENaC. In some embodiments, a therapeutically orprophylactically effective amount of one or more of pharmaceuticalcompositions is administered to a subject in need of such treatment. Insome embodiments, administration of any of the disclosed alpha-ENaC RNAiagents can be used to decrease the number, severity, and/or frequency ofsymptoms of a disease in a subject.

The described pharmaceutical compositions that include an alpha-ENaCRNAi agent can be used to treat at least one symptom in a subject havinga disease or disorder that would benefit from reduction or inhibition inexpression of alpha-ENaC nRNA. In some embodiments, the subject isadministered a therapeutically effective amount of one or morepharmaceutical compositions that include an alpha-ENaC RNAi agentthereby treating the symptom. In other embodiments, the subject isadministered a prophylactically effective amount of one or morealpha-ENaC RNAi agents, thereby preventing or inhibiting the at leastone symptom.

The route of administration is the path by which an alpha-ENaC RNAiagent is brought into contact with the body. In general, methods ofadministering drugs, oligonucleotides, and nucleic acids, for treatmentof a mammal are well known in the art and can be applied toadministration of the compositions described herein. The alpha-ENaC RNAiagents disclosed herein can be administered via any suitable route in apreparation appropriately tailored to the particular route. Thus, insome embodiments, the herein described pharmaceutical compositions areadministered via inhalation, intranasal administration, intratrachealadministration, or oropharyngeal aspiration administration. In someembodiments, the pharmaceutical compositions can be administered byinjection, for example, intravenously, intramuscularly,intracutaneously, subcutaneously, intraarticularly, orintraperitoneally, or topically.

The pharmaceutical compositions including an alpha-ENaC RNAi agentdescribed herein can be delivered to a cell, group of cells, tissue, orsubject using oligonucleotide delivery technologies known in the art. Ingeneral, any suitable method recognized in the art for delivering anucleic acid molecule (in vitro or in vivo) can be adapted for use withthe compositions described herein. For example, delivery can be by localadministration, (e.g., direct injection, implantation, or topicaladministering), systemic administration, or subcutaneous, intravenous,intraperitoneal, or parenteral routes, including intracranial (e.g.,intraventricular, intraparenchymal and intrathecal), intramuscular,transdermal, airway (aerosol), nasal, oral, rectal, or topical(including buccal and sublingual) administration. In some embodiments,the compositions are administered via inhalation, intranasaladministration, oropharyngeal aspiration administration, orintratracheal administration. For example, in some embodiments, it isdesired that the alpha-ENaC RNAi agents described herein inhibit theexpression of an alpha-ENaC gene in the pulmonary epithelium, for whichadministration via inhalation (e.g., by an inhaler device, such as ametered-dose inhaler, or a nebulizer such as a jet or vibrating meshnebulizer, or a soft mist inhaler) is particularly suitable andadvantageous.

In some embodiments, the pharmaceutical compositions described hereincomprise one or more pharmaceutically acceptable excipients. Thepharmaceutical compositions described herein are formulated foradministration to a subject.

As used herein, a pharmaceutical composition or medicament includes apharmacologically effective amount of at least one of the describedtherapeutic compounds and one or more pharmaceutically acceptableexcipients. Pharmaceutically acceptable excipients (excipients) aresubstances other than the Active Pharmaceutical Ingredient (API,therapeutic product, e.g., alpha-ENaC RNAi agent) that are intentionallyincluded in the drug delivery system. Excipients do not exert or are notintended to exert a therapeutic effect at the intended dosage.Excipients can act to a) aid in processing of the drug delivery systemduring manufacture, b) protect, support or enhance stability,bioavailability or patient acceptability of the API, c) assist inproduct identification, and/or d) enhance any other attribute of theoverall safety, effectiveness, of delivery of the API during storage oruse. A pharmaceutically acceptable excipient may or may not be an inertsubstance.

Excipients include, but are not limited to: absorption enhancers,anti-adherents, anti-foaming agents, anti-oxidants, binders, bufferingagents, carriers, coating agents, colors, delivery enhancers, deliverypolymers, detergents, dextran, dextrose, diluents, disintegrants,emulsifiers, extenders, fillers, flavors, glidants, humectants,lubricants, oils, polymers, preservatives, saline, salts, solvents,sugars, surfactants, suspending agents, sustained release matrices,sweeteners, thickening agents, tonicity agents, vehicles,water-repelling agents, and wetting agents.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water-soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, Cremophor®EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol,and sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration can be in theform of a sterile aqueous preparation of the drug that can be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems can also be used to present the drug for bothintra-articular and ophthalmic administration.

Formulations suitable for inhalation administration can be prepared byincorporating the active compound in the desired amount in anappropriate solvent, followed by sterile filtration. In general,formulations for inhalation administration are sterile solutions atphysiological pH and have low viscosity (<5 cP). Salts may be added tothe formulation to balance tonicity. In some cases, surfactants orco-solvents can be added to increase active compound solubility andimprove aerosol characteristics. In some cases, excipients can be addedto control viscosity in order to ensure size and distribution ofnebulized droplets.

The active compounds can be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

The alpha-ENaC RNAi agents can be formulated in compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form refers to physically discrete units suited as unitary dosagesfor the subject to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms of the disclosureare dictated by and directly dependent on the unique characteristics ofthe active compound and the therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals.

A pharmaceutical composition can contain other additional componentscommonly found in pharmaceutical compositions. Such additionalcomponents include, but are not limited to: anti-pruritics, astringents,local anesthetics, or anti-inflammatory agents (e.g., antihistamine,diphenhydramine, etc.). It is also envisioned that cells, tissues, orisolated organs that express or comprise the herein defined RNAi agentsmay be used as “pharmaceutical compositions.” As used herein,“pharmacologically effective amount,” “therapeutically effectiveamount,” or simply “effective amount” refers to that amount of an RNAiagent to produce a pharmacological, therapeutic, or preventive result.

In some embodiments, the methods disclosed herein further comprise thestep of administering a second therapeutic or treatment in addition toadministering an RNAi agent disclosed herein. In some embodiments, thesecond therapeutic is another alpha-ENaC RNAi agent (e.g., an alpha-ENaCRNAi agent that targets a different sequence within the alpha-ENaCtarget). In other embodiments, the second therapeutic can be a smallmolecule drug, an antibody, an antibody fragment, and/or an aptamer.

Generally, an effective amount of an alpha-ENaC RNAi agent disclosedherein will be in the range of from about 0.0001 to about 20 mg/kg ofbody weight/day, e.g., from about 0.001 to about 3 mg/kg of bodyweight/day. In some embodiments, an effective amount of an alpha-ENaCRNAi agent will be in the range of from about 0.001 to about 0.500 mg/kgof body weight per dose. In some embodiments, an effective amount of analpha-ENaC RNAi agent will be in the range of from about 0.001 to about0.100 mg/kg of body weight per dose. In some embodiments, an effectiveamount of an alpha-ENaC RNAi agent will be in the range of from about0.001 to about 0.050 mg/kg of body weight per dose. The amountadministered will also likely depend on such variables as the overallhealth status of the patient, the relative biological efficacy of thecompound delivered, the formulation of the drug, the presence and typesof excipients in the formulation, and the route of administration. Also,it is to be understood that the initial dosage administered can beincreased beyond the above upper level to rapidly achieve the desiredblood-level or tissue level, or the initial dosage can be smaller thanthe optimum.

For treatment of disease or for formation of a medicament or compositionfor treatment of a disease, the pharmaceutical compositions describedherein including an alpha-ENaC RNAi agent can be combined with anexcipient or with a second therapeutic agent or treatment including, butnot limited to: a second or other RNAi agent, a small molecule drug, anantibody, an antibody fragment, peptide, and/or an aptamer.

The described alpha-ENaC RNAi agents, when added to pharmaceuticallyacceptable excipients or adjuvants, can be packaged into kits,containers, packs, or dispensers. The pharmaceutical compositionsdescribed herein can be packaged in dry powder or aerosol inhalers,other metered-dose inhalers, nebulizers, pre-filled syringes, or vials.

Methods of Treatment and Inhibition of Expression

The alpha-ENaC RNAi agents disclosed herein can be used to treat asubject (e.g., a human or other mammal) having a disease or disorderthat would benefit from administration of the RNAi agent. In someembodiments, the RNAi agents disclosed herein can be used to treat asubject (e.g., a human) that would benefit from a reduction and/orinhibition in expression of alpha-ENaC mRNA.

In some embodiments, the RNAi agents disclosed herein can be used totreat a subject (e.g., a human) having a disease or disorder for whichthe subject would benefit from reduction in ENaC channel activity,including but not limited to, for example, cystic fibrosis, chronicbronchitis, non-cystic fibrosis bronchiectasis, chronic obstructivepulmonary disease (COPD), asthma, respiratory tract infections, primaryciliary dyskinesia, and/or lung carcinoma cystic fibrosis and/or dryeye. Treatment of a subject can include therapeutic and/or prophylactictreatment. The subject is administered a therapeutically effectiveamount of any one or more alpha-ENaC RNAi agents described herein. Thesubject can be a human, patient, or human patient. The subject may be anadult, adolescent, child, or infant. Administration of a pharmaceuticalcomposition described herein can be to a human being or animal.

Increased ENaC activity is known to promote airway surface liquiddehydration and impair mucociliary clearance. In some embodiments, thedescribed alpha-ENaC RNAi agents are used to treat at least one symptommediated at least in part by ENaC activity levels, in a subject. Thesubject is administered a therapeutically effective amount of any one ormore of the described alpha-ENaC RNAi agents. In some embodiments, thesubject is administered a prophylactically effective amount of any oneor more of the described RNAi agents, thereby treating the subject bypreventing or inhibiting the at least one symptom.

In certain embodiments, the present disclosure provides methods fortreatment of diseases, disorders, conditions, or pathological statesmediated at least in part by alpha-ENaC gene expression, in a patient inneed thereof, wherein the methods include administering to the patientany of the alpha-ENaC RNAi agents described herein.

In some embodiments, the alpha-ENaC RNAi agents are used to treat ormanage a clinical presentation or pathological state in a subject,wherein the clinical presentation or pathological state is mediated atleast in part by ENaC expression. The subject is administered atherapeutically effective amount of one or more of the alpha-ENaC RNAiagents or alpha-ENaC RNAi agent-containing compositions describedherein. In some embodiments, the method comprises administering acomposition comprising an alpha-ENaC RNAi agent described herein to asubject to be treated.

In some embodiments, the gene expression level and/or mRNA level of analpha-ENaC gene in certain epithelial cells of subject to whom adescribed alpha-ENaC RNAi agent is administered is reduced by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%,relative to the subject prior to being administered the alpha-ENaC RNAiagent or to a subject not receiving the alpha-ENaC RNAi agent. In someembodiments, the ENaC levels or ENaC channel activity levels in certainepithelial cells of a subject to whom a described alpha-ENaC RNAi agentis administered is reduced by at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or greater than 99%, relative to the subject priorto being administered the alpha-ENaC RNAi agent or to a subject notreceiving the alpha-ENaC RNAi agent. The gene expression level, proteinlevel, and/or mRNA level in the subject may be reduced in a cell, groupof cells, and/or tissue of the subject. In some embodiments, thealpha-ENaC mRNA levels in certain epithelial cells subject to whom adescribed alpha-ENaC RNAi agent has been administered is reduced by atleast about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98% relative to the subject prior to being administered thealpha-ENaC RNAi agent or to a subject not receiving the alpha-ENaC RNAiagent. In some embodiments, the level of the ENaC heterotrimeric proteincomplex in certain epithelial cells in a subject to whom a describedalpha-ENaC RNAi agent has been administered is reduced by at least about30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or98% relative to the subject prior to being administered the alpha-ENaCRNAi agent or to a subject not receiving the alpha-ENaC RNAi agent. TheENaC level in the subject may be reduced in a cell, group of cells,tissue, blood, and/or other fluid of the subject. For example, in someembodiments, the level of alpha-ENaC mRNA and/or ENaC heterotrimericprotein complex in pulmonary epithelial cells of a subject to whom adescribed alpha-ENaC RNAi agent has been administered is reduced by atleast about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98% relative to the subject prior to being administered thealpha-ENaC RNAi agent or to a subject not receiving the alpha-ENaC RNAiagent. In some embodiments, the level of alpha-ENaC mRNA and/or ENaCheterotrimeric protein complex and/or ENaC channel activity levels in asubset of pulmonary epithelial cells, such as airway epithelial cells,of a subject to whom a described alpha-ENaC RNAi agent has beenadministered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to the subjectprior to being administered the alpha-ENaC RNAi agent or to a subjectnot receiving the alpha-ENaC RNAi agent.

A reduction in gene expression, mRNA, and protein levels can be assessedby any methods known in the art. Reduction or decrease in alpha-ENaCmRNA level, ENaC channel activity level, and/or ENaC heterotrimericprotein complex levels, are collectively referred to herein as areduction or decrease in alpha-ENaC or inhibiting or reducing theexpression of the alpha-ENaC gene. The Examples set forth hereinillustrate known methods for assessing inhibition of alpha-ENaC geneexpression.

Cells, Tissues, Organs, and Non-Human Organisms

Cells, tissues, organs, and non-human organisms that include at leastone of the alpha-ENaC RNAi agents described herein are contemplated. Thecell, tissue, organ, or non-human organism is made by delivering theRNAi agent to the cell, tissue, organ, or non-human organism.

The above provided embodiments and items are now illustrated with thefollowing, non-limiting examples.

EXAMPLES Example 1. Synthesis of Alpha-ENaC RNAi Agents

The Alpha-ENaC RNAi agent duplexes shown in Table 5 were synthesized inaccordance with the following:

A. Synthesis. The sense and antisense strands of the alpha-ENaC RNAiagents were synthesized according to phosphoramidite technology on solidphase used in oligonucleotide synthesis. Depending on the scale, aMerMade96E® (Bioautomation), a MerMade12® (Bioautomation), or an OPPilot 100 (GE Healthcare) was used. Syntheses were performed on a solidsupport made of controlled pore glass (CPG, 500 Å or 600 Å, obtainedfrom Prime Synthesis, Aston, Pa., USA). All RNA and 2′-modified RNAphosphoramidites were purchased from Thermo Fisher Scientific(Milwaukee, Wis., USA). Specifically, the 2′-O-methyl phosphoramiditesthat were used included the following:(5′-O-dimethoxytrityl-N⁶-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-dimethoxy-trityl-N⁴-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,(5′-O-dimethoxytrityl-N²-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, and5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. The 2′-deoxy-2′-fluoro-phosphoramidites carried thesame protecting groups as the 2′-O-methyl RNA amidites.5′-dimethoxytrityl-2′-O-methyl-inosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites were purchased from Glen Research (Virginia). Theinverted abasic(3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites were purchased from ChemGenes (Wilmington, Mass., USA).The following UNA phosphoramidites were used:5′-(4,4′-Dimethoxytrityl)-N6-(benzoyl)-2′,3′-seco-adenosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,5′-(4,4′-Dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite,5′-(4,4′-Dimethoxytrityl)-N-isobutyryl-2′,3′-seco-guanosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and5′-(4,4′-Dimethoxy-trityl)-2′,3′-seco-uridine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite. TFAaminolink phosphoramidites were also commercially purchased(ThermoFisher).

Tri-alkyne-containing phosphoramidites were dissolved in anhydrousdichloromethane or anhydrous acetonitrile (50 mM), while all otheramidites were dissolved in anhydrous acetonitrile (50 mM) and molecularsieves (3 Å) were added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM inacetonitrile) or 5-Ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile)was used as activator solution. Coupling times were 10 minutes (RNA), 90seconds (2′ 0-Me), and 60 seconds (2′ F). In order to introducephosphorothioate linkages, a 100 mM solution of 3-phenyl1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster,Mass., USA) in anhydrous acetonitrile was employed.

Alternatively, tri-alkyne moieties were introduced post-synthetically(see section E, below). For this route, the sense strand wasfunctionalized with a 5′ and/or 3′ terminal nucleotide containing aprimary amine. TFA aminolink phosphoramidite was dissolved in anhydrousacetonitrile (50 mM) and molecular sieves (3 Å) were added.5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or5-Ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used asactivator solution. Coupling times were 10 minutes (RNA), 90 seconds (2′0-Me), and 60 seconds (2′ F). In order to introduce phosphorothioatelinkages, a 100 mM solution of 3-phenyl 1,2,4-dithiazoline-5-one (POS,obtained from PolyOrg, Inc., Leominster, Mass., USA) in anhydrousacetonitrile was employed.

B. Cleavage and deprotection of support bound oligomer. Afterfinalization of the solid phase synthesis, the dried solid support wastreated with a 1:1 volume solution of 40 wt. % methylamine in water and28% to 31% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30° C.The solution was evaporated and the solid residue was reconstituted inwater (see below).

C. Purification. Crude oligomers were purified by anionic exchange HPLCusing a TSKgel SuperQ-5PW 13 μm column and Shimadzu LC-8 system. BufferA was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile andbuffer B was the same as buffer A with the addition of 1.5 M sodiumchloride. UV traces at 260 nm were recorded. Appropriate fractions werepooled then run on size exclusion HPLC using a GE Healthcare XK 16/40column packed with Sephadex G-25 fine with a running buffer of 100 mMammonium bicarbonate, pH 6.7 and 20% Acetonitrile or filtered water.Alternatively, pooled fractions were desalted and exchanged into anappropriate buffer or solvent system via tangential flow filtration.

D. Annealing. Complementary strands were mixed by combining equimolarRNA solutions (sense and antisense) in 1×PBS (Phosphate-Buffered Saline,1×, Corning, Cellgro) to form the RNAi agents. Some RNAi agents werelyophilized and stored at −15 to −25° C.

Duplex concentration was determined by measuring the solution absorbanceon a UV-Vis spectrometer in 1×PBS. The solution absorbance at 260 nm wasthen multiplied by a conversion factor and the dilution factor todetermine the duplex concentration. Unless otherwise stated, theconversion factor used was 0.037 mg/(mL-cm).

E. Conjugation of Tri-alkyne linker. Either prior to or after annealing,the 5′ or 3′ amine functionalized sense strand is conjugated to atri-alkyne linker. An example tri-alkyne linker structure that can beused in forming the constructs disclosed herein is as follows:

The following describes the conjugation of tri-alkyne linker to theannealed duplex: Amine-functionalized duplex was dissolved in 90%DMSO/10% H₂O, at ˜50-70 mg/mL. 40 equivalents triethylamine was added,followed by 3 equivalents tri-alkyne-PNP. Once complete, the conjugatewas precipitated twice in a solvent system of 1× phosphate bufferedsaline/acetonitrile (1:14 ratio), and dried.

F. Conjugation of Targeting Ligands. Either prior to or after annealing,the 5′ or 3′ tridentate alkyne functionalized sense strand is conjugatedto targeting ligands. The following example describes the conjugation oftargeting ligands to the annealed duplex: Stock solutions of 0.5MTris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5M of Cu(II)sulfate pentahydrate (Cu(II)SO4.5H2O) and 2M solution of sodiumascorbate were prepared in deionized water. A 75 mg/mL solution in DMSOof targeting ligand was made. In a 1.5 mL centrifuge tube containingtri-alkyne functionalized duplex (3 mg, 75 μL, 40 mg/mL in deionizedwater, ˜15,000 g/mol), 25 μL of 1M Hepes pH 8.5 buffer is added. Aftervortexing, 35 μL of DMSO was added and the solution is vortexed.Targeting ligand was added to the reaction (6 equivalents/duplex, 2equivalents/alkyne, ˜15 μL) and the solution is vortexed. Using pHpaper, pH was checked and confirmed to be pH ˜8. In a separate 1.5 mLcentrifuge tube, 50 μL of 0.5M THPTA was mixed with 10 uL of 0.5MCu(II)SO₄.5H₂O, vortexed, and incubated at room temp for 5 min. After 5min, THPTA/Cu solution (7.2 μL, 6 equivalents 5:1 THPTA:Cu) was added tothe reaction vial, and vortexed. Immediately afterwards, 2M ascorbate (5μL, 50 equivalents per duplex, 16.7 per alkyne) was added to thereaction vial and vortexed. Once the reaction was complete (typicallycomplete in 0.5-1 h), the reaction was immediately purified bynon-denaturing anion exchange chromatography.

Example 2. In Vivo Intratracheal Administration of Alpha-ENaC RNAiAgents in Mice

To assess the activity of alpha-ENaC RNAi agents in vivo, male ICR micewere administered 50 microliters via a microsprayer device (PennCentury, Philadelphia, Pa.) suitable for intratracheal (IT)administration on study days 1 and 2, of either isotonic saline vehiclefor use as a control, or 5 mg/kg of one of the following alpha-ENaC RNAiagents without conjugate ligand (i.e., “naked RNAi agent”) formulated inisotonic saline: AD04019, AD04020, AD04021, AD04022, AD04023, AD04024,AD04025, or AD04026. (See, e.g., Tables 3 through 6 for chemicalstructure information for the chemically modified duplexes used in thisExample).

Either 4 or 5 mice were dosed per group. Mice were sacrificed (sac) onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

FIG. 1 shows the relative expression of the identified alpha-ENaC RNAiagent compositions (AD04019, AD04020, AD04021, AD04022, AD04023,AD04024, AD04025, and AD04026), with each RNAi agent showing asignificant reduction in lung alpha-ENaC expression compared to thevehicle control.

Example 3. In Vivo Intratracheal Administration of Alpha-ENaC RNAiAgents in Mice

On study days 1 and 2, male ICR mice were administered 50 microlitersvia a microsprayer device (Penn Century, Philadelphia, Pa.) suitable forintratracheal (IT) administration, of either isotonic saline vehicle touse as a control, or 3 mg/kg of an alpha-ENaC RNAi agent (i.e., eitherAD04025 or AD04858 (see, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplexes used in this Example)),formulated in isotonic saline. Either 4 or 5 mice were dosed per group.Mice were sacrificed on study day 9, and total RNA was isolated fromboth lungs following collection and homogenization. Alpha-ENaC (SCNN1A)mRNA expression was quantitated by probe-based quantitative PCR,normalized to GAPDH expression, and expressed as fraction of vehiclecontrol group (geometric mean, +/−95% confidence interval).

FIG. 2 shows the relative expression of alpha-ENaC RNAi agents AD04025and AD04858, with both RNAi agents showing a significant reduction inlung alpha-ENaC expression compared to control.

Example 4. In Vivo Intratracheal Administration of Alpha-ENaC RNAiAgents with and without Conjugation to Epithelial Cell Targeting Ligandsin Rats

On study days 1 and 2, male Sprague Dawley rats were administered 200microliters via a microsprayer device (Penn Century, Philadelphia, Pa.)suitable for intratracheal (IT) administration, of either 0.5 mg/kg, 1.5mg/kg, or 5 mg/kg of an alpha-ENaC RNAi agent formulated in isotonicsaline. Five (5) rats were dosed per group. Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

FIG. 3 shows the relative expression of alpha-ENaC RNAi agents AD04025and AD04025-conjugate. AD04025-conjugate was synthesized bypost-synthetically linking a peptide-based integrin targeting ligandhaving affinity for αvβ6 integrin, via a masked poly-L-lysine (PLL)scaffold, to an amino group that was added to 5′ terminal end of thesense strand of the RNAi agent. (See, e.g., Tables 3 through 6 forchemical structure information for the chemically modified duplexes usedin this Example). While both the naked RNAi agent and the RNAiagent-conjugate showed a substantial reduction in lung alpha-ENaCexpression compared to baseline measurements, the AD04025-conjugateshowed a numerically improved level of knockdown across each of thethree dosage levels measured (0.5 mg/kg, 1.5 mg/kg, and 5 mg/kg), with aparticularly noticeable improvement at the 1.5 mg/kg dose (78% knockdownwith ligand vs. 47% knockdown without ligand).

Example 5. In Vivo Oropoharyngeal Aspiration Administration ofAlpha-ENaC RNA Agents Conjugated to Epithelial Cell Targeting Ligands inRats

On study day 1, male Sprague Dawley rats were dosed via oropharyngeal(“OP”) aspiration administration with 200 microliters using a pipette,according to the following dosing groups recited in Table 7:

TABLE 7 Dosing Groups of Rats in Example 5 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single OP dose on day 12 0.5 mg/kg of AD05347 conjugated to a tridentate small Single OPmolecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the dose onday 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 3 0.5 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 4 0.5 mg/kg of AD05454 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 5 0.5 mg/kg of AD05455 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 6 0.5 mg/kg of AD05456 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 7 0.5 mg/kg of AD05457 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. (See, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM2 in Groups 2 through 7 has the structurerepresented in FIG. 4, which was conjugated to the RNAi agent via theterminal amine (i.e., by forming a covalent bond with the terminalNH₂—C₆ group) on the 5′ terminal end of the sense strand.

Five (5) rats were dosed per group (n=5). Rats were sacrificed on studyday 9, and total RNA was isolated from both lungs following collectionand homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitatedby probe-based quantitative PCR, normalized to GAPDH expression, andexpressed as fraction of vehicle control group (geometric mean, +/−95%confidence interval).

TABLE 8 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 5 Average Relative rENaC mRNA Expression (n = 5 for Low HighGroup ID each group) (error) (error) Group 1 (isotonic saline) 1.0000.161 0.192 Group 2 (0.5 mg/kg AD05347) 0.411 0.039 0.042 Group 3 (0.5mg/kg AD05453) 0.678 0.092 0.106 Group 4 (0.5 mg/kg AD05454) 0.728 0.1270.154 Group 5 (0.5 mg/kg AD05455) 0.663 0.075 0.084 Group 6 (0.5 mg/kgAD05456) 0.633 0.101 0.120 Group 7 (0.5 mg/kg AD05457) 0.726 0.174 0.228

As shown in Table 8 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control. For example,AD05347, which includes a cyclopropyl-phosphonate group located at the5′ terminal end of the antisense strand, had an average reduction ofapproximately 59% (0.411) of mRNA compared to the control group.

Further, each of the other alpha-ENaC RNAi agents showed a reduction ofat least approximately 2700 of rENaC mRNA compared to control.

Example 6 In Vivo Intratracheal Administration of Alpha-ENaC RNAi AgentsConjugated to Epithelial Cell Targeting Ligands in Rats

On study day 1, male Sprague Dawley rats were administered 200microliters via a microsprayer device (Penn Century, Philadelphia, Pa.)suitable for intratracheal (IT) administration, of either isotonicsaline vehicle for use as a control, or one of the following alpha-ENaCRNAi agents according to the following dosing groups recited in Table 9:

TABLE 9 Dosing Groups of Rats in Example 6 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single IT dose on day 12 1.5 mg/kg of AD04835 conjugated to a tridentate small Single IT dosemolecule αvβ6 epithelial cell targeting ligand (Tri-SM1) at the on day 15′ terminal end of the sense strand, formulated in isotonic saline. 31.5 mg/kg of AD04835 conjugated to a tridentate small Single IT dosemolecule αvβ6 epithelial cell targeting ligand (Tri-SM1) on day 1further including a cysteine-PEG2 linkage at the 5′ terminal end of thesense strand, formulated in isotonic saline. 4 1.5 mg/kg of AD05346conjugated to a tridentate small Single IT dose molecule αvβ6 epithelialcell targeting ligand (Tri-SM1) at the on day 1 5′ terminal end of thesense strand, formulated in isotonic saline. 5 1.5 mg/kg of AD05345conjugated to a tridentate small Single IT dose molecule αvβ6 epithelialcell targeting ligand (Tri-SM1) at the on day 1 5′ terminal end of thesense strand, formulated in isotonic saline. 6 1.5 mg/kg of AD05347conjugated to a tridentate small Single IT dose molecule αvβ6 epithelialcell targeting ligand (Tri-SM1) at the on day 1 5′ terminal end of thesense strand, formulated in isotonic saline. 7 1.5 mg/kg of AD04835conjugated to a monodentate peptide- Single IT dose based αvβ6epithelial cell targeting ligand which further on day 1 included a PEG20linker, followed by a peptide linker (PheCitPhePro (SEQ ID NO: 290)), a20 kilodalton (KDa) PEG group, and a cysteine linker, which was thenconjugated at the 5′ terminal end of the sense strand, formulated inisotonic saline. (See, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM1 in Groups 2, 5, and 6, has the structurerepresented in FIG. 5, which was conjugated to the RNAi agent via theterminal amine (i.e., by forming a covalent bond with the terminalNH₂—C₆ group) on the 5′ terminal end of the sense strand. For Groups 3and 4, the tridentate small molecule ligand in Groups 3 and 4 replacedthe glutaric linker shown in FIG. 5 with a linker that includedcysteine-PEG₂ linkage, represented as follows:

Five (5) rats were dosed in each of Groups 1, 2, 3, 4, 5, and 7 (n=5),and four (4) rats were dosed in Group 6. Rats were sacrificed on studyday 9, and total RNA was isolated from both lungs following collectionand homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitatedby probe-based quantitative PCR, normalized to GAPDH expression, andexpressed as fraction of vehicle control group (geometric mean, +/−95%confidence interval).

TABLE 10 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 6 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.082 0.089 Group 2 (1.5mg/kg AD04835-Tri-SM1) 0.453 0.098 0.126 Group 3 (1.5 mg/kgAD04835-PEG₂-Cys-Tri-SM1) 0.365 0.076 0.095 Group 4 (1.5 mg/kgAD07065-PEG₂-Cys-Tri-SM1) 0.412 0.136 0.204 Group 5 (1.5 mg/kgAD05345-Tri-SM1) 0.404 0.097 0.128 Group 6 (1.5 mg/kg AD05347-Tri-SM1)0.311 0.048 0.057 Group 7 (1.5 mg/kg AD05453-Cys-PEG20 kDa- 0.354 0.0780.101 peptide linker-PEG₂₀-Tri-peptide ligand)

As shown in Table 10 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control. In addition,the use of a tridentate small molecule αvβ6 epithelial cell targetingligand shows comparable reduction in mRNA expression when compared to apeptide-based αvβ6 epithelial cell targeting ligand that furtherincluded a 20 kDa PEG PK modifier.

Example 7. In Vivo Oropharyngeal Aspiration Administration of Alpha-ENaCRNAi Agents Conjugated to Epithelial Cell Targeting Ligands in Rats

On study day 1, male Sprague Dawley rats were dosed via oropharyngeal(“OP”) aspiration administration with 200 microliters using a pipette,according to the following dosing groups recited in Table 11:

TABLE 11 Dosing Groups of Rats in Example 7 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single OP dose on day 12 0.5 mg/kg of AD05347 conjugated to a tridentate small Single OPmolecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the dose onday 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 3 0.5 mg/kg of AD05458 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 4 0.5 mg/kg of AD05459 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 5 0.5 mg/kg of AD05562 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 6 0.5 mg/kg of AD05563 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 7 0.5 mg/kg of AD05564 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) that dose onday 1 further includes a cysteine linking group at the 5′ terminal endof the sense strand, formulated in isotonic saline. 8 0.5 mg/kg ofAD05565 conjugated to a tridentate small Single OP molecule αvβ6epithelial cell targeting ligand (Tri-SM2) at the dose on day 1 5′terminal end of the sense strand, formulated in isotonic saline. 9 0.5mg/kg of AD05567 conjugated to a tridentate small Single OP moleculeαvβ6 epithelial cell targeting ligand (Tri-SM2) at the dose on day 1 5′terminal end of the sense strand, formulated in isotonic saline. 10 0.5mg/kg of AD05570 conjugated to a tridentate small Single OP moleculeαvβ6 epithelial cell targeting ligand (Tri-SM2) at the dose on day 1 5′terminal end of the sense strand, formulated in isotonic saline. (See,e.g., Tables 3 through 6 for chemical structure information for thechemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM2 in each of Groups 2-6 and 8-10, has the structurerepresented in FIG. 4, which was conjugated to the RNAi agent via theterminal amine (i.e., by forming a covalent bond with the terminalNH₂—C₆ group) on the 5′ terminal end of the sense strand. The ligand forGroup 7 included a cysteine linking group (see, e.g., Example 6).

Four (4) rats were dosed in Groups 1, 2, 3, 4, 5, 6, 7, and 9 (n=4), andthree (3) rats were dosed in Groups 8 and 10 (n=3). Rats were sacrificedon study day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 12 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 7 Number Average Relative of rats rENaC mRNA Low High Group ID(n=) Expression (error) (error) Group 1 (isotonic saline) 4 1.000 0.0410.043 Group 2 (0.5 mg/kg AD05347) 4 0.457 0.088 0.109 Group 3 (0.5 mg/kgAD05458) 4 0.708 0.055 0.059 Group 4 (0.5 mg/kg AD05459) 4 0.753 0.1740.227 Group 5 (0.5 mg/kg AD05562) 4 0.608 0.056 0.062 Group 6 (0.5 mg/kgAD05563) 4 0.621 0.048 0.053 Group 7 (0.5 mg/kg AD05564) 4 0.569 0.0950.114 Group 8 (0.5 mg/kg AD05565) 3 0.627 0.066 0.073 Group 9 (0.5 mg/kgAD05567) 4 0.638 0.087 0.100 Group 10 (0.5 mg/kg AD05570) 3 0.645 0.1230.151

As shown in Table 12 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control. For example,AD05347 showed approximately a 54% reduction (0.457) in average rENaCmRNA expression compared to control.

Example 8. In Vivo Oropharyngeal Aspiration Administration of Alpha-ENaCRNAi Agents Conjugated to Epithelial Cell Targeting Ligands in Rats

On study day 1, male Sprague Dawley rats were dosed via oropharyngeal(“OP”) aspiration administration with 200 microliters using a pipette,according to the following dosing groups recited in Table 13:

TABLE 13 Dosing Groups of Rats in Example 8 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single OP dose on day 12 0.5 mg/kg of AD05347 conjugated to a tridentate small Single OPmolecule αvβ6 epithelial cell targeting ligand (Tri-SM2) at the dose onday 1 5′ terminal end of the sense strand, formulated in isotonicsaline. 3 0.5 mg/kg of AD05347 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at thedose on day 1 5′ terminal end of the sense strand, formulated inisotonic saline. 4 0.5 mg/kg of AD05453 conjugated to a tridentate smallSingle OP molecule αvβ6 epithelial cell targeting ligand (Tri-SM2) atthe dose on day 1 5′ terminal end of the sense strand, formulated inisotonic saline. 5 0.5 mg/kg of AD05453 conjugated to a tridentate smallSingle OP molecule αvβ6 epithelial cell targeting ligand (Tri-SM9) atthe dose on day 1 5′ terminal end of the sense strand, formulated inisotonic saline. 6 0.5 mg/kg of AD05453 conjugated to a tridentate smallSingle OP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6) atthe dose on day 1 5′ terminal end of the sense strand, formulated inisotonic saline. 7 0.5 mg/kg of AD05453 conjugated to a tridentate smallSingle OP molecule αvβ6 epithelial cell targeting ligand (Tri-SM8) atthe dose on day 1 5′ terminal end of the sense strand, formulated inisotonic saline. 8 0.5 mg/kg of AD05453 conjugated to a tridentate smallSingle OP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) atthe dose on day 1 5′ terminal end of the sense strand, formulated inisotonic saline. 9 0.5 mg/kg of AD05453 conjugated to a tridentate smallSingle OP molecule αvβ6 epithelial cell targeting ligand (Tri-SM10) atthe dose on day 1 5′ terminal end of the sense strand, formulated inisotonic saline. 10 0.5 mg/kg of AD05453 conjugated to a tridentatesmall Single OP molecule αvβ6 epithelial cell targeting ligand(Tri-SM11) at the dose on day 1 5′ terminal end of the sense strand,formulated in isotonic saline. 11 0.5 mg/kg of AD05453 conjugated to atridentate peptide- Single OP based αvβ6 epithelial cell targetingligand at the 5′ terminal end dose on day 1 of the sense strand,formulated in isotonic saline. (See, e.g., Tables 3 through 6 forchemical structure information for the chemically modified duplexes usedin this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM2 in Group 2 and Group 4 has the structurerepresented in FIG. 4; the tridentate small molecule αvβ6 epithelialcell targeting ligand referred to as Tri-SM6.1 in Groups 3 and 8 has thestructure represented in FIG. 6; the tridentate small molecule αvβ6epithelial cell targeting ligand referred to as Tri-SM9 in Group 5 hasthe structure represented in FIG. 7; the tridentate small molecule αvβ6epithelial cell targeting ligand referred to as Tri-SM6 in Group 6 hasthe structure represented in FIG. 8; the tridentate small molecule αvβ6epithelial cell targeting ligand referred to as Tri-SM8 in Group 7 hasthe structure represented in FIG. 9; the tridentate small molecule αvβ6epithelial cell targeting ligand referred to as Tri-SM10 in Group 9 hasthe structure represented in FIG. 10; and the tridentate small moleculeαvβ6 epithelial cell targeting ligand referred to as Tri-SM11 in Group10 has the structure represented in FIG. 11. Each of the respectivetridentate small molecule αvβ6 epithelial cell targeting ligands wereadded by conjugation via the amino group on the 5′ terminal end of therespective alpha-ENaC RNAi agent.

Four (4) rats were dosed in each Group (n=4). Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 14 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 8 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.162 0.193 Group 2 (0.5mg/kg AD05347-Tri-SM2) 0.469 0.101 0.129 Group 3 (0.5 mg/kgAD05347-Tri-SM6.1) 0.358 0.078 0.100 Group 4 (0.5 mg/kg AD05453-Tri-SM2)0.562 0.086 0.102 Group 5 (0.5 mg/kg AD05453-Tri-SM9) 0.620 0.168 0.230Group 6 (0.5 mg/kg AD05453-Tri-SM6) 0.559 0.099 0.120 Group 7 (0.5 mg/kgAD05453-Tri-SM8) 0.691 0.072 0.081 Group 8 (0.5 mg/kg AD05453-Tri-SM6.1)0.454 0.055 0.063 Group 9 (0.5 mg/kg AD05453-Tri-SM10) 0.454 0.080 0.097Group 10 (0.5 mg/kg AD05453-Tri-SM11) 0.577 0.113 0.140 Group 11 (0.5mg/kg AD05453-tridentate 0.558 0.057 0.064 peptide ligand)

As shown in Table 14 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control. For example,AD05347-Tri-SM6.1 (Group 3) showed approximately a 64% reduction (0.358)in average rENaC mRNA expression compared to control, andAD05453-Tri-SM6.1 (Group 8) showed approximately a 55% reduction (0.454)in average rENaC mRNA expression compared to control. Further, Groups 8and 9 achieved approximately 55% reduction (0.454) in average rENaC mRNAexpression without the use of a 5′ terminal cyclopropyl-phosphonatemodification on the antisense strand, and showed a comparable inhibitoryeffect to Group 2, which had approximately 53% reduction (0.469) inaverage rENaC mRNA expression with 5′ antisense cyclopropyl-phosphonatemodification. Moreover, as observed in groups 4, 6, 8, 9, and 10,tridentate small molecule αvβ6 epithelial cell targeting ligands werecomparable or in some instances numerically superior to Group 11 (e.g.,Groups 8 and 9 that included Tri-SM6.1 and T-SM10), which utilized atridentate peptide-based αvβ6 epithelial cell targeting ligand known tohave affinity for integrin αvβ6 (See International Patent ApplicationPublication No. WO 2018/085415 at FIG. 11 for chemical structureinformation).

Example 9. In Vivo Oropharyngeal Aspiration Administration of Alpha-ENaCRNAi Agents Conjugated to Epithelial Cell Targeting Ligands in Rats

On study day 1, male Sprague Dawley rats were dosed via oropharyngeal(“OP”) aspiration administration with 200 microliters using a pipette,which included the following dosing groups recited in Table 15:

TABLE 15 Dosing Groups of Rats in Example 9 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single OP dose on day 12 0.5 mg/kg of AD05453 without any targeting ligand (i.e., Single OP“naked RNAi agent”), formulated in isotonic saline dose on day 1 3 0.5mg/kg of AD05453 conjugated to a tridentate small Single OP moleculeαvβ6 epithelial cell targeting ligand (Tri-SM6.1) at the dose on day 15′ terminal end of the sense strand, formulated in isotonic saline. 40.5 mg/kg of AD05453 conjugated to a tridentate small Single OP moleculeαvβ6 epithelial cell targeting ligand (Tri-SM7) at the dose on day 1 5′terminal end of the sense strand, formulated in isotonic saline. 5 0.5mg/kg of AD05618 conjugated to a tridentate small Single OP moleculeαvβ6 epithelial cell targeting ligand (Tri-SM6.1) at the dose on day 15′ terminal end of the sense strand, formulated in isotonic saline. 60.5 mg/kg of AD05562 conjugated to a tridentate small Single OP moleculeαvβ6 epithelial cell targeting ligand (Tri-SM6.1) at the dose on day 15′ terminal end of the sense strand, formulated in isotonic saline. 70.5 mg/kg of AD05564 conjugated to a tridentate small Single OP moleculeαvβ6 epithelial cell targeting ligand (Tri-SM6.1) at the dose on day 15′ terminal end of the sense strand, formulated in isotonic saline. 80.5 mg/kg of AD05567 conjugated to a tridentate small Single OP moleculeαvβ6 epithelial cell targeting ligand (Tri-SM6.1) at the dose on day 15′ terminal end of the sense strand, formulated in isotonic saline.(See, e.g., Tables 3 through 6 for chemical structure information forthe chemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM6.1 in Groups 3 and 5-8 has the structurerepresented in FIG. 6.

Four (4) rats were dosed in each Group (n=4). Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 16 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 9 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.00 0.180 0.219 Group 2 (0.5mg/kg AD05453) 0.713 0.139 0.173 Group 3 (0.5 mg/kg AD05453-Tri-SM6.1)0.562 0.082 0.096 Group 4 (0.5 mg/kg AD05453-Tri-SM7) 0.768 0.059 0.064Group 5 (0.5 mg/kg AD05618-Tri-SM6.1) 0.524 0.074 0.086 Group 6 (0.5mg/kg AD05562-Tri-SM6.1) 0.784 0.07 0.077 Group 7 (0.5 mg/kgAD05564-Tri-SM6.1) 0.921 0.104 0.117 Group 8 (0.5 mg/kgAD05567-Tri-SM6.1) 0.707 0.084 0.096

As shown in Table 16 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control. Further, whenadministered naked, AD05453 showed only approximately 29% inhibition(0.713), while when conjugated to Tri-SM6.1 integrin targeting ligand itshowed a 44% reduction (0.562) in average rENaC mRNA expression.

Example 10. In Vivo Oropharyngeal Aspiration Administration ofAlpha-ENaC RNAi Agents Conjugated to Epithelial Cell Targeting Ligandsin Rats

On study day 1, male Sprague Dawley rats were dosed via oropharyngeal(“OP”) aspiration administration with 200 microliters using a pipette,which included the following dosing groups recited in Table 17:

TABLE 17 Dosing Groups of Rats in Example 10 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single OP dose on day 12 0.5 mg/kg of AD05347 conjugated to a tridentate small Single OPmolecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 3 0.5 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 4 0.5 mg/kg of AD05671 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 5 0.5 mg/kg of AD05672 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 6 0.5 mg/kg of AD05673 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 7 0.5 mg/kg of AD05558 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 8 0.5 mg/kg of AD05560 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 9 0.5 mg/kg of AD05611 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 10 0.5 mg/kg of AD05613 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. (See, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM6.1 in Groups 2-10 has the structure represented inFIG. 6.

Four (4) rats were dosed in each Group (n=4). Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 18 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 10 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.084 0.092 Group 2 (0.5mg/kg AD05347-Tri-SM6.1) 0.375 0.128 0.194 Group 3 (0.5 mg/kgAD05453-Tri-SM6.1) 0.597 0.163 0.224 Group 4 (0.5 mg/kgAD05671-Tri-SM6.1) 0.663 0.062 0.068 Group 5 (0.5 mg/kgAD05672-Tri-SM6.1) 0.808 0.114 0.133 Group 6 (0.5 mg/kgAD05673-Tri-SM6.1) 0.623 0.100 0.119 Group 7 (0.5 mg/kgAD05558-Tri-SM6.1) 0.533 0.043 0.047 Group 8 (0.5 mg/kgAD05560-Tri-SM6.1) 0.647 0.122 0.150 Group 9 (0.5 mg/kgAD05611-Tri-SM6.1) 0.477 0.067 0.078 Group 10 (0.5 mg/kgAD05613-Tri-SM6.1) 0.640 0.165 0.223

As shown in Table 18 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control.

Example 1. In Vivo Oropharyngeal Aspiration Administration of Alpha-ENaCRNAi Agents Conjugated to Epithelial Cell Targeting Ligands in Rats

On study day 1, male Sprague Dawley rats were dosed via oropharyngeal(“OP”) aspiration administration with 200 microliters using a pipette,which included the following dosing groups recited in Table 19:

TABLE 19 Dosing Groups of Rats in Example 11 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single OP dose on day 12 0.5 mg/kg of AD05347 conjugated to a tridentate small Single OPmolecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 3 0.5 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 4 0.5 mg/kg of AD05618 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 5 0.5 mg/kg of AD05619 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 6 0.5 mg/kg of AD05622 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 7 0.5 mg/kg of AD05623 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. (See, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM6.1 in Groups 2-7 has the structure represented inFIG. 6.

Five (5) rats were dosed in each Group (n=5). Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 20 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 11 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.195 0.242 Group 2 (0.5mg/kg AD05347-Tri-SM6.1) 0.383 0.041 0.046 Group 3 (0.5 mg/kgAD05453-Tri-SM6.1) 0.489 0.168 0.257 Group 4 (0.5 mg/kgAD05618-Tri-SM6.1) 0.770 0.185 0.244 Group 5 (0.5 mg/kgAD05619-Tri-SM6.1) 0.719 0.080 0.090 Group 6 (0.5 mg/kgAD05622-Tri-SM6.1) 0.564 0.168 0.239 Group 7 (0.5 mg/kgAD05623-Tri-SM6.1) 0.575 0.115 0.144

As shown in Table 20 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control.

Example 12. In Vivo Oropharyngeal Aspiration Administration ofAlpha-ENaC RNAi Agents Conjugated to Epithelial Cell Targeting Ligandsin Rats

On study day 1, male Sprague Dawley rats were dosed via oropharyngeal(“OP”) aspiration administration with 200 microliters using a pipette,according to the following dosing groups recited in Table 21:

TABLE 21 Dosing Groups of Rats in Example 12 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single OP dose on day 12 0.5 mg/kg of AD05347 conjugated to a tridentate small Single OPmolecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 3 0.5 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 4 0.5 mg/kg of AD05683 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 5 0.5 mg/kg of AD05684 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 6 0.5 mg/kg of AD05685 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 7 0.5 mg/kg of AD05686 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 8 0.5 mg/kg of AD05687 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 9 0.5 mg/kg of AD05564 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 10 0.5 mg/kg of AD05688 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 11 0.5 mg/kg of AD05689 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 12 0.5 mg/kg of AD05690 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 13 0.5 mg/kg of AD05691 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. (See, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM6.1 in Groups 2-13 has the structure represented inFIG. 6.

Four (4) rats were dosed in each Group (n=4). Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 22 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 12 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.157 0.186 Group 2 (0.5mg/kg AD05347-Tri-SM6.1) 0.534 0.066 0.075 Group 3 (0.5 mg/kgAD05453-Tri-SM6.1) 0.573 0.086 0.101 Group 4 (0.5 mg/kgAD05683-Tri-SM6.1) 0.547 0.052 0.057 Group 5 (0.5 mg/kgAD05684-Tri-SM6.1) 0.755 0.158 0.200 Group 6 (0.5 mg/kgAD05685-Tri-SM6.1) 0.609 0.077 0.089 Group 7 (0.5 mg/kgAD05686-Tri-SM6.1) 0.591 0.077 0.089 Group 8 (0.5 mg/kgAD05687-Tri-SM6.1) 0.624 0.099 0.118 Group 9 (0.5 mg/kgAD05564-Tri-SM6.1) 0.787 0.172 0.221 Group 10 (0.5 mg/kgAD05688-Tri-SM6.1) 0.563 0.072 0.082 Group 11 (0.5 mg/kgAD05689-Tri-SM6.1) 0.693 0.136 0.169 Group 12 (0.5 mg/kgAD05590-Tri-SM6.1) 0.651 0.159 0.211 Group 13 (0.5 mg/kgAD05691-Tri-SM6.1) 0.870 0.132 0.155

As shown in Table 22 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control.

Example 13. Dose Ranging Study of Oropharyngeal AspirationAdministration of Alpha-ENaC RNAi Agents Conjugated to Epithelial CellTargeting Ligands in Rats

On study day 1, male Sprague Dawley rats were dosed via oropharyngeal(“OP”) aspiration administration with 200 microliters using a pipette,according to the following dosing groups recited in Table 23:

TABLE 23 Dosing Groups of Rats in Example 13 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) Single OP dose on day 12 0.0625 mg/kg of AD05453 conjugated to a tridentate small Single OPmolecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 3 0.125 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 4 0.25 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 5 0.5 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 6 0.75 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 7 1.0 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) at dose onday 1 the 5′ terminal end of the sense strand, formulated in isotonicsaline. 8 3.0 mg/kg of AD05453 conjugated to a tridentate small SingleOP molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1at the doseon day 1 5′ terminal end of the sense strand, formulated in isotonicsaline. (See, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM6.1 in Groups 2-8 has the structure represented inFIG. 6.

Six (6) rats were dosed in each of Groups 1, 2, 3, 4, 7, and 8 (n=5).Four rats were dosed in Groups 5 and 6 (n=4). Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 24 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 13 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.111 0.125 Group 2(0.0625 mg/kg AD05453-Tri-SM6.1) 0.695 0.083 0.095 Group 3 (0.125 mg/kgAD05453-Tri-SM6.1) 0.747 0.139 0.171 Group 4 (0.25 mg/kgAD05453-Tri-SM6.1) 0.631 0.080 0.092 Group 5 (0.5 mg/kgAD05453-Tri-SM6.1) 0.492 0.034 0.037 Group 6 (0.75 mg/kgAD05453-Tri-SM6.1) 0.485 0.113 0.147 Group 7 (1.0 mg/kgAD05453-Tri-SM6.1) 0.433 0.077 0.094 Group 8 (3.0 mg/kgAD05453-Tri-SM6.1) 0.324 0.052 0.062 (See, e.g., Tables 3 through 6 forchemical structure information for the chemically modified duplexes usedin this Example).

As shown in Table 24 above, alpha-ENaC RNAi agent AD05453 showed areduction in mRNA expression in rats compared to control at each of thedosage levels administered.

Example 14. In Vivo Intratracheal Administration of Alpha-ENaC RNAiAgents in Mice

On study days 1 and 2, male ICR mice were administered 50 microlitersvia a microsprayer device (Penn Century, Philadelphia, Pa.) of eitherisotonic saline vehicle for use as a control, or 5 mg/kg of one of thefollowing alpha-ENaC RNAi agents without a conjugate ligand (i.e.,“naked RNAi agent”), formulated in isotonic saline: AD04025, AD04526,AD04527, AD04528, AD04529, AD04530, AD04531, AD04536, or AD04537. 4 micewere dosed per group (n=4). Mice were sacrificed on study day 9, andtotal RNA was isolated from both lungs following collection andhomogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated byprobe-based quantitative PCR, normalized to GAPDH expression, andexpressed as fraction of vehicle control group (geometric mean, +/−95%confidence interval).

TABLE 25 Average Relative mENaC mRNA Expression at Sacrifice (Day 9) inExample 14 Average Relative mENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.117 0.132 Group 2 (0.5mg/kg AD04025) 0.451 0.097 0.123 Group 3 (0.5 mg/kg AD04526) 0.585 0.1080.132 Group 4 (0.5 mg/kg AD04527) 0.403 0.101 0.134 Group 5 (0.5 mg/kgAD04528) 0.498 0.117 0.153 Group 6 (0.5 mg/kg AD04529) 0.480 0.042 0.047Group 7 (0.5 mg/kg AD04530) 0.670 0.006 0.006 Group 8 (0.5 mg/kgAD04531) 0.662 0.103 0.122 Group 9 (0.5 mg/kg AD04536) 0.746 0.101 0.117Group 10 (0.5 mg/kg AD04537) 0.409 0.021 0.022 (See, e.g., Tables 3through 6 for chemical structure information for the chemically modifiedduplexes used in this Example).

As shown in Table 25 above, each of the alpha-ENaC RNAi agents showed areduction in mRNA expression in rats compared to control.

Example 15. In Vivo Intratracheal Administration of Alpha-ENaC RNAiAgents in Mice

On study days 1 and 2, male ICR mice were administered 50 microlitersvia a microsprayer device (Penn Century, Philadelphia, Pa.) of eitherisotonic saline vehicle for use as a control, or 5 mg/kg of one of thefollowing alpha-ENaC RNAi agents without a conjugate ligand (i.e.,“naked RNAi agent”), formulated in isotonic saline: AD04025, AD04538,AD04539, AD04532, AD04533, AD04534, AD04535, or AD04540. (See, e.g.,Tables 3 through 6 for chemical structure information for the chemicallymodified duplexes used in this Example).

Four (4) mice were dosed per group (n=4). Mice were sacrificed on studyday 9, and total RNA was isolated from both lungs following collectionand homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitatedby probe-based quantitative PCR, normalized to GAPDH expression, andexpressed as fraction of vehicle control group (geometric mean, +/−95%confidence interval).

TABLE 26 Average Relative mENaC mRNA Expression at Sacrifice (Day 9) inExample 15 Average Relative mENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.081 0.088 Group 2 (0.5mg/kg AD04025) 0.448 0.097 0.125 Group 3 (0.5 mg/kg AD04538) 0.855 0.1010.115 Group 4 (0.5 mg/kg AD04539) 0.833 0.076 0.083 Group 5 (0.5 mg/kgAD04532) 0.581 0.127 0.162 Group 6 (0.5 mg/kg AD04533) 0.743 0.041 0.044Group 7 (0.5 mg/kg AD04534) 1.006 0.127 0.146 Group 8 (0.5 mg/kgAD04535) 1.042 0.119 0.134 Group 9 (0.5 mg/kg AD04540) 0.982 0.111 0.125(See, e.g., Tables 3 through 6 for chemical structure information forthe chemically modified duplexes used in this Example).

As shown in Table 26 above, the underlying sequence of the respectivealpha-ENaC RNAi agent impacts the level of ENaC gene inhibitionachieved. For example, alpha-ENaC RNAi agent AD04025 includes anantisense strand sequence that is designed to target position 972 of thealpha-ENaC gene (i.e., nucleotides 1-19 of the antisense strand aredesigned to be at least partially complementary to the alpha-ENaC gene(SEQ ID NO:1) at positions 972-990). AD04525 achieved the highest levelof inhibition of the RNAi agents tested in this Example and showedapproximately 55% knockdown of gene expression (0.448) compared tocontrol. The remaining Alpha-ENaC RNAi agents were designed to targetdifferent positions on the gene, including alpha-ENaC RNAi agentsAD04538 (targeting gene position 973), AD04539 (targeting gene position999), AD04532 (targeting gene position 1000), AD04533 (also targetinggene position 973), AD04534 (also targeting gene position 999), AD04535(targeting gene position 1291), and AD04540 (targeting gene position763). As shown above, an alpha-ENaC RNAi agent that is designed totarget the gene at a different position can have different inhibitoryactivity (e.g., compare alpha-ENaC mRNA knockdown levels of AD04025(position 972) with AD04538 (position 973) and AD04533 (position 973)).

Furthermore, when comparing alpha-ENaC RNAi agents at the same position(e.g., AD04539 and AD04534), despite both sequences having underlyingnucleobases designed to inhibit the gene at the same position (e.g.,gene position 999), slight modifications of the underlying base sequenceand/or the inclusion of different modified nucleotides can lead to atleast numerically different inhibition activity.

Example 16. In Vivo Intratracheal Administration of Alpha-ENaC RNAiAgents in Rats

On study days 1 and 2, male Sprague Dawley rats were administered 200microliters via a microsprayer device (Penn Century, Philadelphia, Pa.)of either isotonic saline vehicle for use as a control, or approximately3 mg/kg of one of the following alpha-ENaC RNAi agents without aconjugate ligand (i.e., “naked RNAi agent”), formulated in isotonicsaline: AD04835, AD04022, AD05116, AD05117, AD05118, or AD05119. (See,e.g., Tables 3 through 6 for chemical structure information for thechemically modified duplexes used in this Example).

Five (5) rats were dosed per group (n=5). Rats were sacrificed on studyday 9, and total RNA was isolated from both lungs following collectionand homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitatedby probe-based quantitative PCR, normalized to GAPDH expression, andexpressed as fraction of vehicle control group (geometric mean, +/−95%confidence interval).

TABLE 27 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 16 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.171 0.207 Group 2 (0.5mg/kg AD04835) 0.281 0.043 0.050 Group 3 (0.5 mg/kg AD04022) 0.297 0.0550.067 Group 4 (0.5 mg/kg AD05116) 0.554 0.095 0.115 Group 5 (0.5 mg/kgAD05117) 0.532 0.097 0.119 Group 6 (0.5 mg/kg AD05118) 0.300 0.034 0.038Group 7 (0.5 mg/kg AD05119) 0.496 0.075 0.089 (See, e.g., Tables 3through 6 for chemical structure information for the chemically modifiedduplexes used in this Example).

Table 27, above, provides additional data showing that the underlyingsequence of the respective alpha-ENaC RNAi agent impacts the level ofENaC gene inhibition achieved. For example, alpha-ENaC RNAi agentsAD04025 and AD04835 each include an antisense strand sequence that isdesigned to target position 972 of the alpha-ENaC gene (i.e.,nucleotides 1-19 of the antisense strand are designed to be at leastpartially complementary to the alpha-ENaC gene (SEQ ID NO:1) atpositions 972-990). Of the alpha-ENaC RNAi agents tested in thisExample, these two RNAi agents showed the greatest level of knockdown atgreater than 70%. The remaining Alpha-ENaC RNAi agents were designed totarget different positions on the gene, including alpha-ENaC RNAi agentsAD05116 (targeting gene position 944), AD05117 (targeting gene position945), AD05118 (targeting gene position 1289), and AD05119 (targetinggene position 1579).

Example 17. Multiple Dose, Dose Ranging Study of OropharyngealAspiration Administration of Alpha-ENaC RNAi Agents Conjugated toEpithelial Cell Targeting Ligands in Rats

On study day 1, study day 2, and study day 3, male Sprague Dawley ratswere dosed via oropharyngeal (“OP”) aspiration administration with 200microliters using a pipette, according to the following dosing groupsrecited in Table 28:

TABLE 28 Dosing Groups of Rats in Example 17 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) OP dose on day 1, day2, and day 3 (three total doses) 2 0.005 mg/kg of AD05453 conjugated toa tridentate small OP dose on day 1, molecule αvβ6 epithelial celltargeting ligand (Tri-SM6.1) day 2, and day 3 at the 5′ terminal end ofthe sense strand, formulated in (three total doses) isotonic saline. 30.01 mg/kg of AD05453 conjugated to a tridentate small OP dose on day 1,molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) day 2, andday 3 at the 5′ terminal end of the sense strand, formulated in (threetotal doses) isotonic saline. 4 0.025 mg/kg of AD05453 conjugated to atridentate small OP dose on day 1, molecule αvβ6 epithelial celltargeting ligand (Tri-SM6.1) day 2, and day 3 at the 5′ terminal end ofthe sense strand, formulated in (three total doses) isotonic saline. 50.05 mg/kg of AD05453 conjugated to a tridentate small OP dose on day 1,molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) day 2, andday 3 at the 5′ terminal end of the sense strand, formulated in (threetotal doses) isotonic saline. 6 0.10 mg/kg of AD05453 conjugated to atridentate small OP dose on day 1, molecule αvβ6 epithelial celltargeting ligand (Tri-SM6.1) day 2, and day 3 at the 5′ terminal end ofthe sense strand, formulated in (three total doses) isotonic saline. 70.50 mg/kg of AD05453 conjugated to a tridentate small OP dose on day 1,molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) day 2, andday 3 at the 5′ terminal end of the sense strand, formulated in (threetotal doses) isotonic saline. (See, e.g., Tables 3 through 6 forchemical structure information for the chemically modified duplexes usedin this Example). As noted herein, the same RNAi agent-tridentate smallmolecule αvβ6 epithelial cell targeting ligand conjugate structure(i.e., Tri-SM6.1-AD05453) in this Example may be alternativelysynthesized by using the tri-alkyne functionalized linking group(TriAlk14) as shown in AD05924, instead of post-synthetic addition tothe terminal amino group, as shown in AD05453. (See also Example 1).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM6.1 in Groups 2-7 has the structure represented inFIG. 6.

Seven (7) rats were dosed in each of Groups 1, 2, 3, 4, 5, and 6 (n=7),and six (6) rats were dosed in Group 7 (n=6). Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 29 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 17 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.127 0.146 Group 2(0.005 mg/kg AD05453-Tri-SM6.1) 0.852 0.097 0.109 Group 3 (0.01 mg/kgAD05453-Tri-SM6.1) 0.663 0.103 0.121 Group 4 (0.025 mg/kgAD05453-Tri-SM6.1) 0.589 0.131 0.168 Group 5 (0.05 mg/kgAD05453-Tri-SM6.1) 0.480 0.058 0.066 Group 6 (0.10 mg/kgAD05453-Tri-SM6.1) 0.432 0.056 0.064 Group 7 (0.50 mg/kgAD05453-Tri-SM6.1) 0.279 0.034 0.039

As shown in Table 29 above, alpha-ENaC RNAi agent AD05453 showed areduction in mRNA expression in rats compared to control at each of thedosage levels administered. Further, multiple OP dose administrationshowed signs of further knockdown of rENaC mRNA expression compared tosingle dose when using the same alpha-ENaC RNAi agent (compare, e.g.,Group 7 of Example 17 with Group 5 of Example 13).

Example 18. In Vivo Intratracheal Administration of Alpha-ENaC RNAiAgents in Mice and Human COPD Sputum Stability Assessment

To assess and compare the activity and stability of a known prior artduplex to the RNAi agents disclosed herein, a duplex having thefollowing modified structure, as disclosed International PatentApplication Publication No: WO 2008/152131 to Novartis et al. (see Table1C therein at ND-9201), was synthesized:

Antisense strand sequence (5′→3′): (SEQ ID NO: 291)GAUUUGUUCUGGUUGcAcAdTsdT Sense strand sequence (5′→3′): (SEQ ID NO: 292) uGuGcAAccAGAAcAAAucdTsdT(hereinafter referred to as ND-9201). According to WO 2008/152131,ND-9201 showed comparatively potent in vitro inhibition of alpha-ENaCgene expression.

First, studies were conducted to assess alpha-ENaC inhibition activityin vivo. On study days 1 and 2, male ICR mice were administered via amicrosprayer device (Penn Century, Philadelphia, Pa.) either isotonicglucose (D5W) vehicle for use as a control, or approximately 10 mg/kg ofND-9201 formulated in D5W. Mice were sacrificed on day 9, and total RNAwas isolated from both lungs following collection and homogenization.Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-basedquantitative PCR, normalized to GAPDH expression, and expressed asfraction of vehicle control group. For comparison, on study days 1 and2, male ICR mice were administered via a microsprayer device (PennCentury, Philadelphia, Pa.) either D5W vehicle for use as a control, orapproximately 5 mg/kg of the RNAi agent AD04025 disclosed hereinformulated in D5W. (See, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplex of AD04025). Mice weresimilarly sacrificed on day 9, and total RNA was isolated from bothlungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNAexpression was quantitated by probe-based quantitative PCR, normalizedto GAPDH expression, and expressed as fraction of vehicle control group.

For ND-9201, at 10 mg/kg dosing on days 1 and 2, approximately 25%inhibition of mENaC mRNA expression was achieved in mice in vivo.

For AD04025, at only 5 mg/kg dosing on days 1 and 2, approximately 65%inhibition of mENaC mRNA expression was achieved in mice in vivo, thusshowing a substantial improvement in inhibition activity over the knownprior art duplex ND-9201.

Additionally, stability studies were conducted with ND-9201 and AD04858in human sputum taken from patients diagnosed with COPD (See, e.g.,Tables 3 through 6 for chemical structure information for the chemicallymodified duplex of AD04858). A solution containing 50 μL of sputum and350 μL of lysis buffer was vortexed, and 12.5 μL of either ND-9201 orAD04858 was added and briefly vortexed each hour. LCMS was conducted onthe samples to determine the remaining full-length product of both thesense strand and the antisense strand of each of the molecules overtime. After 6 hours, AD04858 showed improved stability, as it hadapproximately 20 to 30% greater full-length product present for both thesense strand and the antisense strand.

Example 19. In Vivo Study of Oropharyngeal Aspiration Administration ofAlpha-ENaC RNAi Agents Conjugated to Epithelial Cell Targeting Ligandsin Rats

On study day 1 and study day 2, male Sprague Dawley rats were dosed viaoropharyngeal (“OP”) aspiration administration with 200 microlitersusing a pipette, which included the following dosing groups recited inTable 30:

TABLE 30 Dosing Groups of Rats in Example 19 Group RNAi Agent and DoseDosing Regimen 1 Isotonic saline (no RNAi agent) OP dose on day 1 andday 2 2 0.025 mg/kg of AD05625 conjugated to a tridentate small OP doseon day 1 molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) andday 2 at the 5′ terminal end of the sense strand, formulated in isotonicsaline. 3 0.50 mg/kg of AD05453 conjugated to a tridentate small OP doseon day 1 molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) andday 2 at the 5′ terminal end of the sense strand, formulated in isotonicsaline. 4 0.50 mg/kg of AD05829 conjugated to a tridentate small OP doseon day 1 molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) andday 2 at the 5′ terminal end of the sense strand, formulated in isotonicsaline. 5 0.50 mg/kg of AD05831 conjugated to a tridentate small OP doseon day 1 molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) andday 2 at the 5′ terminal end of the sense strand, formulated in isotonicsaline. 6 0.50 mg/kg of AD05833 conjugated to a tridentate small OP doseon day 1 molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1) andday 2 at the 5′ terminal end of the sense strand, formulated in isotonicsaline. (See, e.g., Tables 3 through 6 for chemical structureinformation for the chemically modified duplexes used in this Example).

The tridentate small molecule αvβ6 epithelial cell targeting ligandreferred to as Tri-SM6.1 in Groups 2-7 has the structure represented inFIG. 6.

Four (4) rats were dosed in each Group (n=7). Rats were sacrificed onstudy day 9, and total RNA was isolated from both lungs followingcollection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression wasquantitated by probe-based quantitative PCR, normalized to GAPDHexpression, and expressed as fraction of vehicle control group(geometric mean, +/−95% confidence interval).

TABLE 31 Average Relative rENaC mRNA Expression at Sacrifice (Day 9) inExample 19 Average Relative rENaC mRNA Low High Group ID Expression(error) (error) Group 1 (isotonic saline) 1.000 0.196 0.243 Group 2(0.25 mg/kg AD05625-Tri-SM6.1) 0.663 0.107 0.127 Group 3 (0.50 mg/kgAD05453-Tri-SM6.1) 0.490 0.091 0.111 Group 4 (0.50 mg/kgAD05829-Tri-SM6.1) 0.767 0.163 0.207 Group 5 (0.50 mg/kgAD05831-Tri-SM6.1) 0.542 0.113 0.142 Group 6 (0.50 mg/kgAD05833-Tri-SM6.1) 0.599 0.025 0.026

In Table 31 above, alpha-ENaC RNAi agents AD05625 and AD05453 eachincluded an antisense strand that was designed to target the alpha-ENaCgene beginning at position 972 (see SEQ ID NO:1); AD05829 included anantisense strand that was designed to target the alpha-ENaC genebeginning at position 944; AD05831 included an antisense strand that wasdesigned to target the alpha-ENaC gene beginning at position 973; andAD01289 included an antisense strand that was designed to target thealpha-ENaC gene beginning at position 1289.

Each of the alpha-ENaC RNAi agents showed inhibition of gene expression,with RNAi agent AD05453 showing comparatively potent inhibition ofalpha-ENaC.

Example 20. In Vivo Topical Ocular Administration of Alpha-ENaC RNAiAgents in Mice

To evaluate the ability of alpha-ENaC RNAi agents to inhibit expressionof alpha ENaC mRNA in the ocular surface epithelium, CB57B1/6 mice(n=3/group) received twice daily topical ocular instillations of salinevehicle or 400 micrograms AD04858 (in two microliter volume) in botheyes for five days. (See, e.g., Tables 3 through 6 for chemicalstructure information for the chemically modified duplex of AD04858). Onstudy day five, mice were sacrificed, samples of the conjunctivalepithelium collected and total RNA isolated from tissue homogenate.Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-basedquantitative PCR, normalized to GAPDH expression, and expressed asfraction of vehicle control group.

After five days of twice daily topical dosing of AD04858, conjunctivalsamples from treated mice expressed significantly less (approximately24%) alpha ENaC mRNA than samples from vehicle treated controls

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An RNAi agent for inhibiting expression of an alpha-ENaC gene,comprising: an antisense strand comprising at least 17 contiguousnucleotides differing by 0 or 1 nucleotides from any one of thesequences provided in Table 2 or Table 3; and a sense strand comprisinga nucleotide sequence that is at least partially complementary to theantisense strand.
 2. The RNAi agent of claim 1, wherein the antisensestrand comprises nucleotides 2-18 of any one of the sequences providedin Table 2 or Table
 3. 3. The RNAi agent of claim 1, wherein the sensestrand comprises a nucleotide sequence of at least 17 contiguousnucleotides differing by 0 or 1 nucleotides from any one of thesequences provided in Table 2 or Table 4, and wherein the sense strandhas a region of at least 85% complementarity over the 17 contiguousnucleotides to the antisense strand.
 4. The RNAi agent of claim 1,wherein at least one nucleotide of the alpha-ENaC RNAi agent is amodified nucleotide or includes a modified internucleoside linkage. 5.The RNAi agent of claim 1, wherein all or substantially all of thenucleotides are modified nucleotides.
 6. The RNAi agent of claim 4,wherein the modified nucleotide is selected from the group consistingof: 2′-O-methyl nucleotide, 2′-fluoro nucleotide, 2′-deoxy nucleotide,2′,3′-seco nucleotide mimic, locked nucleotide, 2′-F-arabino nucleotide,2′-methoxyethyl nucleotide, abasic nucleotide, ribitol, invertednucleotide, inverted 2′-O-methyl nucleotide, inverted 2′-deoxynucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide,morpholino nucleotide, vinyl phosphonate deoxyribonucleotide, and3′-O-methyl nucleotide.
 7. The RNAi agent of claim 5, wherein all orsubstantially all of the nucleotides are modified with either2′-O-methyl nucleotides or 2′-fluoro nucleotides.
 8. The RNAi agent ofclaim 1, wherein the antisense strand comprises the nucleotide sequenceof any one of the modified sequences provided in Table
 3. 9. The RNAiagent of claim 1, wherein the sense strand comprises the nucleotidesequence of any one of the modified sequences provided in Table
 4. 10.The RNAi agent of claim 1, wherein the antisense strand comprises thenucleotide sequence of any one of the modified sequences provided inTable 3 and the sense strand comprises the nucleotide sequence of anyone of the modified sequences provided in Table
 4. 11. The RNAi agent ofclaim 1, wherein the RNAi agent is linked to a targeting ligand.
 12. TheRNAi agent of claim 11, wherein the targeting ligand comprises anintegrin targeting ligand.
 13. The RNAi agent of claim 12, wherein theintegrin targeting ligand is an αvβ6 integrin targeting ligand.
 14. TheRNAi agent of claim 13, wherein the αvβ6 integrin targeting ligand hasthe structure represented by any one of the structures of FIGS. 4-11.15. The RNAi agent of claim 11, wherein the targeting ligand isconjugated to the sense strand.
 16. The RNAi agent of claim 15, whereinthe targeting ligand is conjugated to the 5′ terminal end of the sensestrand.
 17. The RNAi agent of claim 1, wherein the sense strand isbetween 18 and 30 nucleotides in length, and the antisense strand isbetween 18 and 30 nucleotides in length.
 18. The RNAi agent of claim 17,wherein the sense strand and the antisense strand are each between 18and 27 nucleotides in length.
 19. The RNAi agent of claim 18, whereinthe sense strand and the antisense strand are each between 18 and 24nucleotides in length.
 20. The RNAi agent of claim 19, wherein the sensestrand and the antisense strand are each 21 nucleotides in length. 21.The RNAi agent of claim 20, wherein the RNAi agent has two blunt ends.22. The RNAi agent of claim 1, wherein the sense strand comprises one ortwo terminal caps.
 23. The RNAi agent of claim 22, wherein the sensestrand comprises one or two inverted abasic residues.
 24. The RNAi agentof claim 1, wherein the RNAi agent is comprised of a sense strand and anantisense strand that form a duplex having the structure of any one ofthe duplexes in Table
 5. 25. The RNAi agent of claim 1, wherein the RNAiagent is comprised of a sense strand and an antisense strand, whereinthe antisense strand comprises a modified nucleotide sequence thatdiffers by 0 or 1 nucleotides from one of the following nucleotidesequences (5′→3′): (SEQ ID NO: 2) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg;(SEQ ID NO: 6) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc); (SEQ ID NO: 10)cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg; (SEQ ID NO: 107)usGfsasUfuUfgUfuCfuGfgUfuGfcAfcAfsg;  or (SEQ ID NO: 152)asGfsasAfgUfcAfuUfcUfgCfuCfuGfcusu; 

wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; srepresents a phosphorothioate linkage, cPrpu represents a 5′-cyclopropylphosphonate-2′-O-methyl uridine; and wherein all or substantially all ofthe nucleotides on the sense strand are modified nucleotides.
 26. TheRNAi agent of claim 25, wherein the sense strand further includes anabasic residue at the 3′ terminal end.
 27. The RNAi agent of claim 1,wherein the RNAi agent comprises an antisense strand and a sense strand,wherein the antisense strand and the sense strand comprise a nucleotidesequence pair selected from the group consisting of: SEQ ID NOs:2 and 4;SEQ ID NOs: 3 and 5; SEQ ID NOs: 6 and 8; SEQ ID NOs: 7 and 9; and SEQID NOs: 10 and
 4. 28. The RNAi agent of claim 1, wherein the RNAi agenthas the duplex structure selected from the group consisting of: AD05453,AD05625, AD05347, AD05831, AD05833, AD04835, and AD05924.
 29. The RNAiagent of claim 1, wherein the RNAi agent includes an antisense strandand a sense strand, wherein the antisense strand and the sense strandconsist of, consist essentially of, or comprise nucleotide sequencesthat differ by 0 or 1 nucleotides from one of the following nucleotidesequence (5′→3′) pairs: (SEQ ID NO: 3) UAUUUGUUCUGGUUGCACAGG and(SEQ ID NO: 5) CCUGUGCAACCAGAACAAAUA; (SEQ ID NO: 7)UAUUUGUUCUGGUUGCACAGC and (SEQ ID NO: 9) GCUGUGCAACCAGAACAAAUA;(SEQ ID NO: 230) UGAUUUGUUCUGGUUGCACAG and (SEQ ID NO: 259)CUGUGCAACCAGAACAAAUCA; or (SEQ ID NO: 254) AGAAGUCAUUCUGCUCUGCUU and(SEQ ID NO: 289) GCAGAGCAGAAUGACUUCUUU.


30. The RNAi agent of claim 29, wherein all or substantially all of thenucleotides are modified nucleotides.
 31. The RNAi agent of claim 29,wherein the sense strand of the RNAi agent is linked to targetingligand.
 32. The RNAi agent of claim 31, wherein the targeting ligand hasaffinity for a cell receptor expressed on an epithelial cell.
 33. TheRNAi agent of claim 31, wherein the targeting ligand is an αvβ6 integrintargeting ligand.
 34. A composition comprising the RNAi agent of claim1, wherein the composition comprises a pharmaceutically acceptableexcipient.
 35. The composition of claim 34, further comprising a secondRNAi agent for inhibiting the expression of alpha-ENaC.
 36. Thecomposition of claim 34, further comprising one or more additionaltherapeutics.
 37. The composition of claim 34, wherein the compositionis formulated for administration by inhalation.
 38. The composition ofclaim 37, wherein the composition is delivered by a metered-doseinhaler, jet nebulizer, vibrating mesh nebulizer, or soft mist inhaler.39. A method for inhibiting expression of an alpha-ENaC gene in a cell,the method comprising introducing into a cell an effective amount of anRNAi agent of claim
 1. 40. The method of claim 39, wherein the cell iswithin a subject.
 41. The method of claim 40, wherein the subject is ahuman subject.
 42. The method of claim 39, wherein the alpha-ENaC geneexpression is inhibited by at least about 30%.
 43. A method forinhibiting expression of an alpha-ENaC gene in a cell, the methodcomprising introducing into a cell an effective amount of thecomposition of claim
 34. 44. The method of claim 43, wherein the cell iswithin a subject.
 45. The method of claim 44, wherein the subject is ahuman subject.
 46. The method of claim 43, wherein the alpha-ENaC geneexpression is inhibited by at least about 30%.
 47. A method of treatingone or more symptoms or diseases associated with enhanced or elevatedENaC activity levels, the method comprising administering to a humansubject in need thereof a therapeutically effective amount of thecomposition of claim
 34. 48. The method of claim 47, wherein the diseaseis a respiratory disease.
 49. The method of claim 48, wherein therespiratory disease is cystic fibrosis, chronic bronchitis, non-cysticfibrosis bronchiectasis, chronic obstructive pulmonary disease (COPD),asthma, respiratory tract infections, primary ciliary dyskinesia, orlung carcinoma cystic fibrosis.
 50. The method of claim 47, wherein thedisease is an ocular disease, such as dry eye syndrome.
 51. The methodof claim 44, wherein the RNAi agent is administered at a dose of about0.001 mg/kg to about 0.500 mg/kg of body weight.
 52. The method of claim51, wherein the RNAi agent is administered in two or more doses.
 53. Themethod of claim 47, wherein the RNAi agent is administered at a dose ofabout 0.001 mg/kg to about 0.500 mg/kg of body weight.
 54. The method ofclaim 53, wherein the RNAi agent is administered in two or more doses.